Recombinant derivatives of botulinum neurotoxins engineered for trafficking studies and neuronal delivery

ABSTRACT

This invention relates to isolated  Clostridium botulinum  propeptides and neurotoxins, isolated nucleic acid molecules encoding  Clostridium botulinum  propeptides and neurotoxins, methods of expression, treatment methods, and methods of detecting neurotoxin trafficking. The isolated  Clostridium botulinum  propeptides have a light chain region; a heavy chain region, where the light and heavy chain regions are linked by a disulfide bond; an intermediate region connecting the light and heavy chain regions and comprising a highly specific protease cleavage site; and an S6 peptide sequence according to SEQ ID NO:2 positioned upstream from, but not attached directly to, the N-terminus of the neurotoxin propeptide at the light chain region to enable site specific attachment of cargo.

This application claims benefit of U.S. Provisional Patent ApplicationSer. No. 61/298,078, filed Jan. 25, 2010, which is hereby incorporatedby reference in its entirety.

This invention was made with government support under NIH-NIAIDAI072466, NIH NINDS NS050276, NIH-NCRR RR017990, and NIH Office of theDirector DP2-OD004631. The government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates to isolated Clostridium botulinum propeptides andneurotoxins, isolated nucleic acid molecules encoding Clostridiumbotulinum propeptides and neurotoxins, methods of expression, treatmentmethods, methods of detecting neurotoxin trafficking, and methods ofdetecting levels of neuronal activity.

BACKGROUND OF THE INVENTION

Botulinum neurotoxins (“BoNTs”) are a family of structurally similarproteins that cause peripheral neuromuscular blockade and respiratoryparalysis. BoNTs are exceedingly toxic, possessing an extremely low LD₅₀(1-50 ng/kg) (National Institute of Occupational Safety and Health,Registry of Toxic Effects of Chemical Substances (R-TECS), Cincinnati,Ohio: National Institute of Occupational Safety and Health, 1996). Thereare seven major BoNT serotypes (BoNT A-G) and multiple subtypes (Smithet al., “Sequence Variation Within Botulinum Neurotoxin SerotypesImpacts Antibody Binding and Neutralization,” Infect. Immun.73(9):5450-5457 (2005)), but all have common structural features and asimilar mechanism of action. BoNTs are synthesized as single chainpropeptides with intramolecular disulfide bonds (Mr approximately150,000; approximately 1,300 amino acids) with extensive areas ofsequence homology. The majority are activated by proteolytic cleavage togenerate a disulfide-bonded heterodimer containing light (approximately50 kDa) and heavy (approximately 100 kDa) chains (“LC” and “HC”respectively).

Botulinum neurotoxin serotype A (“BoNT/A”) heterodimer has beenextensively studied and has been found to contain three functionaldomains. Toxicity is associated with metalloprotease activity confinedto the LC, neuron binding activity is associated with the C-terminalhalf of the HC (HC_(C)), and translocation activity responsible fordelivering the LC protease to the neuronal cytosol is associated withthe N-terminal half of the HC (HC_(N)) (Johnson, “Clostridial Toxins asTherapeutic Agents: Benefits of Nature's Most Toxic Proteins,” Annu.Rev. Microbiol. 53:551-575 (1999); Montecucco et al., “Structure andFunction of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys.28(4):423-472 (1995)).

The toxicity of BoNTs is a consequence of a multi-step mechanismculminating in a LC-mediated proteolytic event that disrupts theneuronal machinery for synaptic vesicle exocytosis. During BoNTpoisoning, BoNTs must first cross epithelial barriers by transcytosis(Simpson, “Identification of the Major Steps in Botulinum Toxin Action,”Annu. Rev. Pharmacol. Toxicol. 44:167-193 (2004)). The BoNT then passesinto the circulation by an unknown pathway, from which it selectivelytargets the presynaptic membrane of motor neurons at neuromuscularjunctions. Toxicity at the neuromuscular junction involves (i) bindingto the plasma membrane, (ii) internalization into endocytic vesicles,(iii) activation within an acidic endosomal compartment that enablesHC-mediated translocation of the LC into the neuronal cytoplasm, and(iv) catalytic cleavage by the LC zinc-endopeptidase of proteincomponents in the neuronal machinery required for synaptic vesicleexocytosis.

The endopeptidase activity responsible for toxicity is associated with aHExxHxxH (SEQ ID NO:1) motif in the LC which is characteristic of thethermolysin family of metalloproteases. Mutagenesis experiments with theBoNT/A light chain have identified the minimal essential domain fortoxicity (Kurazono et al., “Minimal Essential Domains SpecifyingToxicity of the Light Chains of Tetanus Toxin and Botulinum NeurotoxinType A,” J. Biol. Chem. 267(21):14721-14729 (1992)), and have pinpointedthe amino acids involved in Zn²⁺ coordination at the metalloproteaseactive site (Rigoni et al., “Site-Directed Mutagenesis IdentifiesActive-Site Residues of the Light Chain of Botulinum Neurotoxin Type A,”Biochem. Biophys. Res. Commun. 288(5):1231-1237 (2001)). These data arecorroborated by crystallography-based structures currently available forthe majority of BoNT serotypes, and by crystallographic data for LC/LCmutants expressed as single entities, or co-crystallized with thesubstrate or inhibitors (Lacy et al., “Crystal Structure of BotulinumNeurotoxin Type A and Implications for Toxicity,” Nat. Struct. Biol.5(10):898-902 (1998); Breidenbach et al., “Substrate RecognitionStrategy for Botulinum Neurotoxin Serotype A,” Nature 432:925-929(2004); Fu et al., “Light Chain of Botulinum Neurotoxin Serotype A:Structural Resolution of a Catalytic Intermediate,” Biochemistry45:8903-8911 (2006); Garcia-Rodriguez et al., “Molecular Evolution ofAntibody Cross-Reactivity for Two Subtypes of Type A BotulinumNeurotoxin,” Nat. Biotechnol. 25:107-116 (2007); Burnett et al.,“Inhibition of Metalloprotease Botulinum Serotype A From aPseudo-Peptide Binding Mode to a Small Molecule That is Active inPrimary Neurons,” J. Biol. Chem. 282:5004-5014 (2007); Silvaggi et al.,“Structures of Clostridium botulinum Neurotoxin Serotype A Light ChainComplexed with Small-Molecule Inhibitors Highlight Active-SiteFlexibility,” Chem. Biol. 14(5):533-542 (2007); Silvaggi et al.,“Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealedby High Resolution Structure of an Inhibitory Peptide Complex,”Biochemistry 47(21):5736-5745 (2008); Zuniga et al., “A PotentPeptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A Has a VeryDifferent Conformation than SNAP-25 Substrate,” Structure 16:588-1597(2008); Kumaran et al., “Structure- and Substrate-Based Inhibitor Designfor Clostridium botulinum Neurotoxin Serotype A,” J. Biol. Chem.283:18883-18891 (2008); Kumaran et al., “Substrate Binding Mode and ItsImplication on Drug Design for Botulinum Neurotoxin A,” PloS Pathog.4(9):e1000165 (2008); Swaminathan et al., “Structural Analysis of theCatalytic and Binding Sites of Clostridium botulinum Neurotoxin B,” Nat.Struct. Biol. 7(8):693-699 (2000); Hanson et al., “Cocrystal Structureof Synaptobrevin-II Bound to Botulinum Neurotoxin Type B at 2.0 ÅResolution,” Nat. Struct. Biol. 7(8):687-692 (2000); Eswaramoorthy etal., “Novel Mechanism for Clostridium botulinum Neurotoxin Inhibition,”Biochemistry 41:9795-9802 (2002); Eswaramoorthy et al., “Role of MetalsIn the Biological Activity of Clostridium botulinum Neurotoxins,”Biochemistry 43(8):2209-2216 (2004); Jin et al., “Structural andBiochemical Studies of Botulinum Neurotoxin Serotype C1 Light ChainProtease: Implications for Dual Substrate Specificity,” Biochemistry46:10685-10693 (2007); Arndt et al., “Structure of Botulinum NeurotoxinType D Light Chain at 1.65 Å Resolution: Repercussions for VAMP-2Substrate Specificity,” Biochemistry 45:3255-3262 (2006); Agarwal etal., “Structural Analysis of Botulinum Neurotoxin Type E CatalyticDomain and Its Mutant Glu₂₁₂>Gln Reveals the Pivotal Role of the Glu₂₁₂Carboxylate in the Catalytic Pathway,” Biochemistry 43:6637-6644 (2004);Agarwal et al., “Analysis of Active Site Residues of BotulinumNeurotoxin E By Mutational, Functional, and Structural Studies:Glu₃₃₅>Gln is an Apoenzyme,” Biochemistry 44:8291-8302 (2005); Agarwalet al., “SNAP-25 Substrate Peptide (Residues 180-183) Binds to ButBypasses Cleavage by Catalytically Active Clostridium botulinumNeurotoxin E,” J. Biol. Chem. 283:25944-25951 (2008); Agarwal et al.,“Structural Analysis of Botulinum Neurotoxin Serotype F Light Chain:Implications on Substrate Binding and Inhibitor Design,” Biochemistry44:11758-11765 (2005); Agarwal et al., “Mode of VAMP SubstrateRecognition and Inhibition of Clostridium botulinum Neurotoxin F,” Nat.Struct. Mol. Biol. 16:789-794 (2009); Arndt et al., “Crystal Structureof Botulinum Neurotoxin Type G Light Chain: Serotype Divergence InSubstrate Recognition,” Biochemistry 44:9574-9580 (2005)).

Recombinant BoNT proteins or peptides have been reported for severalserotypes, primarily as part of efforts aimed at developing a vaccineagainst BoNT poisoning. The receptor-binding HC domain (HC_(C)) has beenproduced in a variety of expression systems. These recombinant HCpreparations were effective immunogens and protected animals challengedwith wt BoNTs (Byrne et al., “Development of Vaccines for Prevention ofBotulism,” Biochimie 82:955-966 (2000); Ravichandran et al., “TrivalentVaccine Against Botulinum Toxin Serotypes A, B, and E That Can BeAdministered By the Mucosal Route,” Infect. Immun. 75(6):3043-3054(2007); Baldwin et al., “Subunit Vaccine Against the Seven Serotypes ofBotulism,” Infect. Immun. 76(3):1314-131 (2008); Smith, “Development ofRecombinant Vaccines for Botulinum Neurotoxin,” Toxicon 36:1539-1548(1998); Baldwin et al., “Characterization of the Antibody Response tothe Receptor Binding Domain of Botulinum Neurotoxin Serotypes A and E,”Infect Immun. 73(10):6998-7005 (2005); Woodward et al., “Expression ofHC Subunits from Clostridium botulinum Types C and D and TheirEvaluation as Candidate Vaccine Antigens In Mice,” Infect. Immun.71(5):2941-2944 (2003); Webb et al., “Protection with RecombinantClostridium botulinum C1 and D Binding Domain Subunit (Hc) VaccinesAgainst C and D Neurotoxins,” Vaccine 25(21):4273-4282 (2007); Lee etal., “C-Terminal Half Fragment (50 kDa) of Heavy Chain Components ofClostridium botulinum Type C and D Neurotoxins Can Be Used As anEffective Vaccine,” Microbiol. Immunol. 51(4):445-455 (2007);LaPenotiere et al., “Expression of a Large, Nontoxic Fragment ofBotulinum Neurotoxin Serotype A and Its Use As an Immunogen,” Toxicon33(10):1383-1386 (1995); Clayton et al., “Protective Vaccination with aRecombinant Fragment of Clostridium botulinum Neurotoxin Serotype AExpressed From A Synthetic Gene In Escherichia coli,” Infect. Immun.63(7):2738-2742 (1995); Byrne et al., “Purification, Potency, andEfficacy of the Botulinum Neurotoxin Type A Binding Domain from Pichiapastoris As a Recombinant Vaccine Candidate,” Infect Immun.66(10):4817-4822 (1998); Lee et al., “Candidate Vaccine AgainstBotulinum Neurotoxin Serotype A Derived From a Venezuelan EquineEncephalitis Virus Vector System,” Infect. Immun. 69(9):5709-5715(2001); Maddaloni et al., “Mucosal Vaccine Targeting Improves Onset ofMucosal and Systemic Immunity to Botulinum Neurotoxin A,” J. Immunol.177(8):5524-5532 (2006); Yu et al., “The Recombinant He Subunit ofClostridium botulinum Neurotoxin Serotype A Is an Effective BotulismVaccine Candidate,” Vaccine 27(21):2816-2822 (2009); Boles et al.,“Recombinant C Fragment of Botulinum Neurotoxin B Serotype (rBoNTB (HC))Immune Response and Protection In the Rhesus Monkey,” Toxicon47(8):877-884 (2006); Zeng et al., “Protective Immunity Against BotulismProvided By a Single Dose Vaccination With an Adenovirus-VectoredVaccine,” Vaccine 25(43):7540-7548 (2007); Xu et al., “An AdenoviralVector-Based Mucosal Vaccine Is Effective In Protection AgainstBotulism,” Gene Ther. 16(3):367-375 (2009); Byrne et al., “Fermentation,Purification, and Efficacy of a Recombinant Vaccine Candidate AgainstBotulinum Neurotoxin Type F From Pichia pastoris,” Protein Expr. Purif.18(3):327-337 (2000); Holley et al., “Cloning, Expression and Evaluationof a Recombinant Sub-Unit Vaccine Against Clostridium botulinum Type FToxin,” Vaccine 19(2-3):288-297 (2000); Foynes et al., “VaccinationAgainst Type F Botulinum Toxin using Attenuated Salmonella enterica vartyphimurium Strains Expressing the BoNT/F H(C) Fragment,” Vaccine21(11-12):1052-1059 (2003); Yu et al., “Evaluation of a Recombinant Heof Clostridium botulinum Neurotoxin Serotype F As an Effective SubunitVaccine,” Clin. Vaccine Immunol. 15(12):1819-1823 (2008)). RecombinantHC_(C) was additionally demonstrated to retain the ability totranscytose epithelia, thereby providing effective immunogen delivery byinhalation (Baldwin et al., “Subunit Vaccine Against the Seven Serotypesof Botulism,” Infect. Immun. 76(3):1314-131 (2008)). Enzymaticallyactive and inactive recombinant LC derivatives have also been expressed(Kurazono et al., “Minimal Essential Domains Specifying Toxicity of theLight Chains of Tetanus Toxin and Botulinum Neurotoxin Type A,” J. Biol.Chem. 267(21):14721-14729 (1992); Rigoni et al., “Site-DirectedMutagenesis Identifies Active-Site Residues of the Light Chain ofBotulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun.288(5):1231-1237 (2001); Breidenbach et al., “Substrate RecognitionStrategy for Botulinum Neurotoxin Serotype A,” Nature 432:925-929(2004); Fu et al., “Light Chain of Botulinum Neurotoxin Serotype A:Structural Resolution of a Catalytic Intermediate,” Biochemistry45:8903-8911 (2006); Silvaggi et al., “Structures of Clostridiumbotulinum Neurotoxin Serotype A Light Chain Complexed withSmall-Molecule Inhibitors Highlight Active-Site Flexibility,” Chem.Biol. 14(5):533-542 (2007); Kumaran et al., “Structure- andSubstrate-Based Inhibitor Design for Clostridium botulinum NeurotoxinSerotype A,” J. Biol. Chem. 283:18883-18891 (2008); Zhou et al.,“Expression and Purification of the Light Chain of Botulinum NeurotoxinA: A Single Mutation Abolishes Its Cleavage of SNAP-25 and NeurotoxicityAfter Reconstitution With the Heavy Chain,” Biochemistry34(46):15175-15181 (1995); Li et al., “High-Level Expression,Purification, and Characterization of Recombinant Type A BotulinumNeurotoxin Light Chain,” Protein Expr. Purif. 17(3):339-344 (1999);Kadkhodayan et al., “Cloning, Expression, and One-Step Purification ofthe Minimal Essential Domain of the Light Chain of Botulinum NeurotoxinType A,” Protein Expr. Purif. 19(1):125-130 (2000); Ahmed et al., “LightChain of Botulinum A Neurotoxin Expressed As an Inclusion Body From aSynthetic Gene Is Catalytically and Functionally Active,” J. ProteinChem. 19(6):475-487 (2000); Li et al., “Probing the Mechanistic Role ofGlutamate Residue In the Zinc-Binding Motif of Type A BotulinumNeurotoxin Light Chain,” Biochemistry 39(9):2399-2405 (2000); Ahmed etal., “Enzymatic Autocatalysis of Botulinum A Neurotoxin Light Chain,” J.Protein Chem. 20(3):221-231 (2001); Ahmed et al., “Factors AffectingAutocatalysis of Botulinum A Neurotoxin Light Chain,” Protein J.23(7):445-451 (2004); Segelke et al., “Crystal Structure of Clostridiumbotulinum Neurotoxin Protease In a Product-Bound State: Evidence forNoncanonical Zinc Protease Activity,” Proc. Natl. Acad. Sci. (USA)101(18):6888-6893 (2004); Baldwin et al., “The C-Terminus of BotulinumNeurotoxin Type A Light Chain Contributes to Solubility, Catalysis, andStability,” Protein Expr. Purif. 37(1):187-195 (2004); Ahmed et al.,“Identification of Residues Surrounding the Active Site of Type ABotulinum Neurotoxin Important for Substrate Recognition and CatalyticActivity,” Protein J. 27(3):151-162 (2008)). These have been found to benon-toxic in vivo even when LC enzymatic activity was preserved, becausethe presence of disulfide-bonded HC is required for BoNT targeting. TheLC expressed as a separate entity, or as part of a holotoxoid, is lessimmunogenic than HC (Smith et al., “Sequence Variation Within BotulinumNeurotoxin Serotypes Impacts Antibody Binding and Neutralization,”Infect. Immun. 73(9):5450-5457 (2005)).

To achieve LC conformations that more closely resemble the native toxin,and to generate a greater variety of antigens for vaccine design, theenzymatically active endopeptidase constructs representing LC fused tothe full and C-terminally truncated version of HC_(N) were alsoexpressed in E. coli (Chaddock et al., “Expression and Purification ofCatalytically Active, Non-Toxic Endopeptidase Derivatives of Clostridiumbotulinum Toxin Type A,” Protein Expr. Purif. 25(2):219-228 (2002);Jensen et al., “Expression, Purification, and Efficacy of the Type ABotulinum Neurotoxin Catalytic Domain Fused to Two Translocation DomainVariants,” Toxicon 41(6):691-701 (2003); Sutton et al., “Preparation ofSpecifically Activatable Endopeptidase Derivatives of Clostridiumbotulinum Toxins Type A, B, and C and Their Applications,” Protein Expr.Purif. 40(1):31-41 (2005)). With subsequent improvements in theconstructs and the expression system, these derivatives were used asbuilding blocks to re-target specificity of botulinum neurotoxin Athrough substitution of native HC_(C) with wheat germ agglutinin (WGA),NGF, and EGF (Chaddock et al., “Inhibition of Vesicular Secretion InBoth Neuronal and Nonneuronal Cells By a Retargeted EndopeptidaseDerivative of Clostridium botulinum Neurotoxin Type A,” Infect. Immun.68(5):2587-2593 (2000); Chaddock et al., “A Conjugate Composed of NerveGrowth Factor Coupled to a Non-Toxic Derivative of Clostridium botulinumNeurotoxin Type A Can Inhibit Neurotransmitter Release In vitro,” GrowthFactors 18(2):147-155 (2000); Duggan et al., “Inhibition of Release ofNeurotransmitters From Rat Dorsal Root Ganglia By a Novel Conjugate of aClostridium botulinum Toxin A Endopeptidase Fragment and Erythrinacristagalli Lectin,” J. Biol. Chem. 277(38):34846-34852 (2002); Chaddocket al., “Retargeted Clostridial Endopeptidases: Inhibition ofNociceptive Neurotransmitter Release In vitro, and AntinociceptiveActivity In In vivo Models of Pain,” Mov. Disord. Suppl 8:S42-S47(2004); Foster et al., “Re-Engineering the Target Specificity ofClostridial Neurotoxins—A Route to Novel Therapeutics,” Neurotox. Res.9(2-3):101-107 (2006)).

Several laboratories have reported expressing recombinant, full-lengthBoNTs in E. coli. Rummel et al., “Two Carbohydrate Binding Sites in theHC_(C)-Domain of Tetanus Neurotoxin Are Required for Toxicity,” J. Mol.Biol. 326(3):835-847 (2003); Rummel et al., “The HC_(C)-Domain ofBotulinum Neurotoxins A and B Exhibits a Singular Ganglioside BindingSite Displaying Serotype Specific Carbohydrate Interaction,” Mol.Microbiol. 51(3):631-643 (2004); Rummel et al., “Synaptotagmins I and IIAct as Nerve Cell Receptors for Botulinum Neurotoxin G,” J. Biol. Chem.279(29):30865-30870 (2004); and Bade et al., “Botulinum Neurotoxin TypeD Enables Cytosolic Delivery of Enzymatically Active Cargo Proteins toNeurons Via Unfolded Translocation Intermediates,” J. Neurochem.91(6):1461-1472 (2004), described the expression of full-length singlechain BoNT/G, /D, /B, and /A in E. coli, either as the wt, or with athrombin-specific cleavage site inserted between the HC and LC, or withthe LC protease inactivated by a point mutation. Kiyatkin et al.,“Induction of an Immune Response by Oral Administration of RecombinantBotulinum Toxin,” Infect. Immun. 65:4586-4591 (1997), reported theexpression of BoNT/C in E. coli, with three inactivating point mutations(H₂₂₉>G; E₂₃₀>T; H₂₃₃>N) in the LC protease, without the insertion ofany specific proteolytic cleavage site between the LC and HC. There wasno evidence that this BoNT/C single chain was processed into adisulfide-bonded heterodimer in vivo, but it was effective as animmunogen when orally administered. In all reports, the single chainholotoxin expressed in E. coli was not secreted into the culture mediumor periplasm and had to be recovered from whole cell lysates. Expressionproblems in E. coli are associated with improper protein foldingstemming from the reducing environment in the E. coli cytosol, thetendency of E. coli to segregate unfolded recombinant proteins withinaggregates of inclusion bodies, proteolytic degradation, and a strongcodon bias against AT-rich clostridial genes.

Recently, a recombinant, atoxic BoNT/A holotoxoid was expressed in thenon-toxic strain of Clostridium botulinum, LNT01, with a yield ofapproximately 1 mg/L (Pier et al., “Recombinant Holotoxoid VaccineAgainst Botulism,” Infect. Immun. 76(1):437-442 (2008)). Thisrecombinant holotoxoid had the mutations R₃₆₄>A and Y₃₆₆>F introducedinto the LC (BoNT/A^(RYM)), and lacked the ability to cleave thesubstrate SNAP 25 in vitro. Mice were challenged with up to 1 μg of thisderivative (approximately 3.3×10⁴ mouse LD₅₀) and monitored for 96hours. All mice survived challenge with 1 μg of single-chain ortrypsin-nicked dichain of BoNT/A^(RYM). Immunization with thisholotoxoid effectively protected mice against lethal BoNT/A challenge.Although this report is encouraging, no information has yet beenprovided regarding the physiological trafficking of BoNT/A^(RYM) incomparison with wt BoNT/A.

The most recent report describes the production of catalyticallyinactive BoNT/A holoprotein (H₂₂₃>A; E₂₂₄>A; H₂₂₇>A) in P. pastoris(ciBoNT/A HP) (Webb et al., “Production of Catalytically InactiveBoNT/A1 Holoprotein and Comparison With BoNT/A1 Subunit Vaccines AgainstToxin Subtypes A1, A2, and A3,” Vaccine 27(33):4490-4497 (2009)). Theprotein expressed from the synthetic gene, which was optimized for codonbias in the host, accumulated intracellularly. There was no introductionof an artificial cleavage site into the loop between LC and HC in thepropeptide. The protein was purified in several steps with conventionalion exchange chromatographies. The yield of highly purified product wasreported to be approximately 1 milligram from four grams of the frozenmethylotrophic yeast. ciBoNT/A HP provided excellent protectiveimmunity, not only against the homologous toxin, but also against twodistinct toxin subtypes with significant amino acid divergence. Micechallenged with 50 μg of this derivative (approximately 1.7×10⁶ mouseLD₅₀) and monitored for 240 hours did not display discernible signs ofBoNT intoxication.

The selectivity of BoNT targeting to neurons has led severallaboratories to consider using BoNT-based molecular vehicles fordelivering therapeutic agents. Early work reported that the HC and LC ofwt BoNTs could be separated, and that the wt HC could be reconstitutedin vitro with either wt LC, or with recombinant LC which could carrypoint mutations, such as His₂₂₇>Tyr, which rendered the LC atoxic (Zhouet al., “Expression and Purification of the Light Chain of BotulinumNeurotoxin A: A Single Mutation Abolishes Its Cleavage of SNAP-25 andNeurotoxicity After Reconstitution With the Heavy Chain,” Biochemistry34(46):15175-15181 (1995); Maisey et al., “Involvement of theConstituent Chains of Botulinum Neurotoxins A and B In the Blockade ofNeurotransmitter Release,” Eur. J. Biochem. 177(3):683-691 (1988);Sathyamoorthy et al., “Separation, Purification, PartialCharacterization and Comparison of the Heavy and Light Chains ofBotulinum Neurotoxin Types A, B, and E,” J. Biol. Chem.260(19):10461-10466 (1985)). The reconstituted BoNT holotoxinderivatives had a severely reduced ability to transport LC into theneuronal cytosol, probably resulting from the harsh conditions requiredfor HC-LC separation and the difficulty of renaturing the protein andreconstituting native disulfide bonds. Attempts have also been made touse isolated wt HC for targeted delivery, by chemically coupling dextranto the HC to provide sites for attaching fluorescent markers ortherapeutic agents (Goodnough et al., “Development of a Delivery Vehiclefor Intracellular Transport of Botulinum Neurotoxin Antagonists,” FEBSLett. 513:163-168 (2002)). Although this “semi-synthetic” BoNTderivative was internalized by neurons, the dextran remained localizedto the endosomal compartment and the specificity of the uptake wasuncertain. Direct chemical or biochemical attachment of cargo moleculesto the HC of BoNTs may not be sufficient for achieving cytosolicdelivery, because structural features associated with the toxin LC arerequired for translocation to the cytosol (Baldwin et al., “TheC-Terminus of Botulinum Neurotoxin Type A Light Chain Contributes toSolubility, Catalysis, and Stability,” Protein Expr. Purif.37(1):187-195 (2004); Brunger et al., “Botulinum Neurotoxin Heavy ChainBelt as an Intramolecular Chaperone for the Light Chain,” PLoS Pathog.3(9):e113 (2007)). Moreover, when chemical methods are used to attachcargo to BoNT toxoids, cargo attachment is not sufficiently selectiveand, consequently, produces a heterogeneous population of derivatives.These problems limit the utility of chemically labeled BoNTs as probesfor definitive demonstration of BoNT trafficking pathways.

Bade et al., “Botulinum Neurotoxin Type D Enables Cytosolic Delivery ofEnzymatically Active Cargo Proteins to Neurons Via UnfoldedTranslocation Intermediates,” J. Neurochem. 91(6):1461-1472 (2004),described recombinant full-length derivatives of BoNT/D as effectivedelivery vehicles which were expressed in E. coli with or without aninactivating mutation (E₂₃₀>A) to the LC protease. To evaluate thedelivery of prototypic cargo proteins in neuronal cultures, greenfluorescent protein (“GFP”), dihydrofolate reductase, fireflyluciferase, or BoNT/A LC were fused to the amino terminus of therecombinant BoNT/D holotoxin. Delivery to the cytosol was evaluated bymeasuring cleavage of the BoNT/D cytoplasmic substrate, synaptobrevin.Dihydrofolate reductase and BoNT/A LC were reported to be effectivelydelivered. When luciferase or GFP were the cargo, delivery of thecorresponding BoNT/D LC catalytic activity to the cytosol wassignificantly reduced, presumably due to the large size of the cargo(luciferase) or its rigidity (GFP) (Brejc et al., “Structural Basis forDual Excitation and Photoisomerization of the Aequorea victoria GreenFluorescent Protein,” Proc. Natl. Acad. Sci. (USA) 94(6):2306-1231(1997); Palm et al., “The Structural Basis for Spectral Variations inGreen Fluorescent Protein,” Nat. Struct. Biol. 4(5):361-365 (1997)).

It has proven particularly difficult to successfully engineertranslocation of recombinant toxin LCs from an endosomal compartment tothe cytosol. This translocation requires acidification of the lumenalmilieu, either to trigger a conformational change in the BoNTheterodimer or to enable its interaction with a translocation mediator(Brunger et al., “Botulinum Neurotoxin Heavy Chain Belt as anIntramolecular Chaperone for the Light Chain,” PLoS Pathog. 3(9):e113(2007); Kamata et al., “Involvement of Phospholipids In the IntoxicationMechanism of Botulinum Neurotoxin,” Biochim. Biophys. Acta.1199(1):65-68 (1994); Tortorella et al., “Immunochemical Analysis of theStructure of Diphtheria Toxin Shows all Three Domains Undergo StructuralChanges at Low pH,” J. Biol. Chem. 270(46):27439-27445 (1995);Tortorella et al., “Immunochemical Analysis Shows All Three Domains ofDiphtheria Toxin Penetrate Across Model Membranes,” J. Biol. Chem.270(46):27446-27452 (1995)). A requirement for cooperation between theBoNT LC and the translocation domain of the HC is supported by evidencedemonstrating that a decapeptide motif, common to the HC_(N) of severalBoNT serotypes as well as to diphtheria and anthrax toxins, is requiredfor successful translocation of the LC to the cytosol (Ratts et al., “AConserved Motif in Transmembrane Helix 1 of Diphtheria Toxin MediatesCatalytic Domain Delivery to the Cytosol,” Proc. Natl. Acad. Sci. (USA)102(43):15635-15640 (2005)). Future development of BoNTs as carriervehicles will require a deeper understanding of how the LC itself, andits interaction with HC_(N), contributes to this mechanism.

Although efforts to express recombinant BoNTs have succeeded inproducing effective immunogens, which in some cases are competent forepithelial transcytosis, these efforts have not produced recombinantproteins with the structural features required for targeting theneuronal cytosol with the efficiency of wt toxins. These limitationsemphasize the importance of selecting an expression system capable ofproducing full-length BoNT derivatives that retain native toxinstructure, disulfide bonding, and physiological trafficking. Also, workfrom multiple laboratories has clarified how the structural domains ofwt botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, butimportant questions remain unanswered. Because BoNT/A intoxicationdisables its own uptake, wt light chain does not accumulate in neuronsat detectable levels.

The present invention is directed to overcoming these and otherlimitations in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to an isolated Clostridiumbotulinum neurotoxin propeptide. The propeptide has a light chainregion; a heavy chain region, where the light and heavy chain regionsare linked by a disulfide bond; an intermediate region connecting thelight and heavy chain regions and comprising a highly specific proteasecleavage site which has three or more specific adjacent amino acidresidues that are recognized by the highly specific protease in order toenable cleavage; and a peptide sequence to enable site-specificattachment of cargo, where the peptide sequence is positioned upstreamof the light chain region and is separated from the N-terminus of thelight chain region by an amino acid spacer sequence.

Another aspect of the present invention relates to an isolated nucleicacid molecule encoding the Clostridium botulinum neurotoxin propeptideof the present invention as well as expression systems and host cellscontaining this nucleic acid molecule.

A further aspect of the present invention relates to an isolated,physiologically active Clostridium botulinum neurotoxin produced bycleaving the Clostridium botulinum neurotoxin propeptide of the presentinvention. The propeptide is cleaved at the highly specific proteasecleavage site. The light and heavy chain regions are linked by adisulfide bond.

Yet another aspect of the present invention relates to a method ofexpressing a recombinant physiologically active Clostridium botulinumneurotoxin. This method involves providing a nucleic acid constructhaving a nucleic acid molecule encoding a Clostridium botulinumneurotoxin propeptide of the present invention. The nucleic acidconstruct has a heterologous promoter operably linked to the nucleicacid molecule and a 3′ regulatory region operably linked to the nucleicacid molecule. The nucleic acid construct is introduced into a host cellunder conditions effective to express the physiologically activeClostridium botulinum neurotoxin.

A further aspect of the present invention relates to a treatment method.This method involves providing the isolated Clostridium botulinumneurotoxin of the present invention, where the cargo comprises atherapeutic agent and administering the isolated Clostridium botulinumneurotoxin to an individual in need of treatment under conditionseffective to provide treatment to the individual.

Another aspect of the present invention relates to a method of detectingClostridium botulinum neurotoxin trafficking. This method involvesexpressing a recombinant physiologically active Clostridium botulinumneurotoxin as described herein. A fluorophore is coupled to theneurotoxin. Trafficking of the neurotoxin is then detected by detectingone or more locations of the fluorophore.

A further aspect of the present invention relates to a method ofdetecting levels of neuronal activity. This method involves providingthe isolated Clostridium botulinum neurotoxin of the present inventionand administering the neurotoxin to an individual or a tissue sample.The method further involves detecting location of the neurotoxin, wheredetection of the neurotoxin at a specific site in the individual ortissue sample indicates an increased level of activity of neurons atthat site.

The invention described herein relates to the design, expression, andpurification of recombinant, full length, BoNT heterodimers that retainall key structural elements required for native BoNT trafficking.Moreover, the expression constructs have been designed to contain ashort peptide sequence that enables site selective attachment of cargomolecules using mild enzymatic conditions that do not contribute toprotein denaturation. Because the BoNT derivatives of the presentinvention contain point mutations that inactivate the LC protease, theywill be capable of accumulating in neurons at higher levels than wtBoNT, which will improve their efficiency for trafficking studies, cargodelivery, detection of neuronal activity, and therapeutic use. Usingsmall molecule fluorophores as prototypic cargo, these derivativesprovide unique molecular tools for studying the sequential steps in BoNTtranslocation and targeting events, and for defining the limits of thepotential cargo that can be delivered to the neuronal cytoplasm with theuse of this system for site-specific cargo attachment.

A series of BoNT/A derivatives have been designed, expressed, andpurified that retain the wild type features required for nativetrafficking. For example, BoNT/A1ad^(ek) and BoNT/A1ad^(tev) are fulllength derivatives rendered atoxic through double point mutations in theLC protease (E₂₂₄>A and Y₃₆₆>A). ΔLC-peptide -BoNT/A^(tev) andΔLC-GFP-BoNT/A^(tev) are derivatives where the catalytic portion of theLC is replaced with a short peptide or with GFP plus the peptide. In allfour of these derivatives, the S6 peptide sequence GDSLSWLLRLLN (SEQ IDNO:2) has been fused to the N-terminus of the proteins to enablesite-specific attachment of cargo using Sfp phosphopantetheinyltransferase. Cargo can be attached in a manner that provides ahomogeneous derivative population rather than a polydisperse mixture ofsingly and multiply-labeled molecular species. All four of theseexemplary derivatives contain an introduced cleavage site for conversioninto disulfide-bonded heterodimers. These constructs were expressed in abaculovirus system and the proteins were secreted into culture mediumand purified to homogeneity in yields ranging from 1 to 30 mg per liter.Derivatives of the present invention provide unique tools to study toxintrafficking in vivo, and to assess how the structure of cargo linked tothe heavy chain influences delivery to the neuronal cytosol. Moreover,they enable engineering of BoNT-based molecular vehicles that can targettherapeutic agents to the neuronal cytoplasm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an amino acid sequence alignment showing features of expressedBoNT/A derivatives, including BoNT/Aad^(ek) (SEQ ID NO:3),BoNT/Aad^(tev) (SEQ ID NO:4), ΔLC-Peptide-BoNT/A^(tev) (SEQ ID NO:5),and ΔLC-GFP-BoNT/A^(tev) (SEQ ID NO:6), according to several embodimentsof the present invention, in comparison with wild type BoNT A (GenBankAccession No. ABP48106 (SEQ ID NO:7)). Space between arrowheads andarrow tails represent regions of sequence identity omitted forsimplicity. Numbers in the upper row correspond to amino acid residuesin wt BoNT/A. Residues that are identical in all proteins are shown inregular type. Introduced mutations are shown in italics. Added aminoacids are shown in bold. A signal peptide for insect cell secretion ofthe expressed derivatives into medium is indicated by “SP”. Tags usedfor affinity chromatography are indicated: polyhistidine tag isindicated by “10 His”; StrepTag II is indicated by “StrepTag II”. “TEV”and the amino acid sequence on light shading represent tobacco etchvirus protease recognition (TEV) sequence. Amino acids on dark shadingrepresent an enterokinase recognition sequence. “S6” identifies apeptide tag used for site-specific attachment of cargo to the expressedproteins. “GFP” represents a portion of green fluorescent protein. Thefive proteins are aligned to illustrate homology between respectivestructural domains. Gaps have been introduced to facilitate thealignment. Spaces between rectangle-enclosed sequences represent sitesof proteolytic cleavage. The disulfide bridge between residues of thelight and heavy chains are shown as long horizontal brackets.

FIGS. 2A-B show BoNT/Aad^(ek) propeptide purification. A reduced 12% SDSPAGE was stained with Coomassie BB R-250. FIG. 2A: TALON® chromatographypurification: lane 1, wt BoNT/A, control; lane 2, unfractionated sampleof concentrated and dialyzed Sf-900 II medium containing secretedpropeptide BoNT/Aad^(ek) prior to loading on column; lane 3, column flowthrough; lane 4, wash 1, loading buffer; lane 5, wash 2, loading bufferwith 20 mM imidazole; lane 6, elution, loading buffer with 200 mMimidazole. FIG. 2B: StrepTactin agarose chromatography: lane 1, sampleof concentrated and dialyzed fraction from FIG. 2A, lane 6 prior toloading on column; lane 2, flow through; lanes 3-7, washes with loadingbuffer; lanes 8-12, elutions with loading buffer and 3 mM desthiobiotin;lane 13, wt BoNT/A, control.

FIGS. 3A-B show processing of BoNT/Aad^(ek) propeptide to heterodimer byproteolytic cleavage with recombinant enterokinase (rEK). One microgramof BoNT/Aad^(ek) propeptide per lane was treated with 0-5 U rEK at 16°C. for 12 hours, separated by 12% SDS PAGE, and stained with CoomassieBB R-250. FIG. 3A: non-reduced samples. FIG. 3B: samples reduced byaddition of β-mercaptoethanol. Lane 1, no rEK; lane 2, 0.001 U rEK; lane3, 0.01 U rEK; lane 4, 0.1 U rEK; lane 5, 1 U rEK; lane 6, 5 U rEK; lane7, wt BoNT/A, control.

FIG. 4 shows removal of 10-His tag from BoNT/Aad^(ek) propeptide bytreatment with AcTEV. BoNT/Aad^(ek) was either treated with buffer (oddlane numbers) or AcTEV protease (even lane numbers; 1 U per microgram,30° C.; see Examples infra for details) for the times indicated: lanes 1and 2, one hour; lanes 3 and 4, two hours; lanes 5 and 6, three hours;lanes 7 and 8, four hours; lanes 9 and 10, six hours. Samples wereloaded on the 12% SDS PAGE in the presence of β-mercaptoethanol,separated, and transferred to nitrocellulose. Western blot was probedwith HRP-coupled anti-His MAb (Santa Cruz, H-3 His probe, Cat #sc-8036HRP). The low MW band (approximately 30 kDa) in even lanes representsAcTEV protease which was supplied by Invitrogen as a 6-His taggedrecombinant enzyme.

FIGS. 5A-B show in vitro fluorescent labeling of AcTEV-treated andrEK-treated BoNT/Aad^(ek) with Sfp phosphopantetheinyl transferase andCoA 547. Lanes 1-4, unreduced samples; lanes 5-8, samples reduced byaddition of β-mercaptoethanol. Lanes 1, 3, 5, 7: 0.02 μg BoNT/Aad^(ek);lanes 2, 4, 6, 8: 0.1 μg BoNT/Aad^(ek). FIG. 5A: 10.5-14% Criterion gel(Bio-Rad) stained with Bio-Safe Coomassie (Bio-Rad). FIG. 5B: Westernblot of gel shown in FIG. 5A scanned on a Typhoon 9500 scanner (GEHealthcare) using 300V PMT, 532/580 nm excitation/emission filter set(green).

FIG. 6 is an ESI Q-TOF MS/MS spectrum of the C-terminal tryptic peptideisolated from enterokinase-processed BoNT/Aad^(ek) LC under reducingconditions. The b and y ion series have been included at the top of FIG.6 to identify the peptide fragment peaks found in the spectrum, wherethe position of vertical bars separating each amino acid(s) correspondto position of the respective ion m/z within the spectrum. Only the mostintense peaks from y series have been labeled for simplicity.

FIGS. 7A-C are MALDI mass spectra and ESI Q-TOF MS/MS spectra of thetryptic dipeptide with internal disulfide bridge linking light and heavychains of enterokinase-processed BoNT/Aad^(ek). FIG. 7A is a MALDI-TOFmass spectrum of an in-gel tryptic digest of the enterokinase-processedBoNT/Aad^(ek) LC, separated and isolated from SDS PAGE run in thepresence of DTT. FIG. 7B is a MALDI-TOF mass spectrum of an in-geltryptic digest of the enterokinase-processed BoNT/Aad^(ek) separated andisolated from SDS PAGE run without reducing agent. A peak at m/z 1489.84matched the predicted m/z of the dipeptide with internal disulfidebridge. FIG. 7C is an ESI Q-TOF MS/MS spectrum of the dipeptide with m/z1489.8 shown in FIG. 7B, confirming presence of the S—S bond in thedipeptide. The b and y ion series have been included at the top of thepanel to identify the peptide fragment peaks found in the spectrum,where the position of vertical bars separating each amino acid(s)correspond to position of the respective ion m/z within the spectrum.Only the most intense peaks have been labeled for clarity.

FIGS. 8A-C show BoNT/Aad^(tev), ΔLC-Peptide-BoNT/A^(tev), and ΔLC-GFP-BoNT/A^(tev) expressed in the baculovirus system, purified by metalchelate and StrepTactin affinity chromatography, and processed withAcTEV. Lanes 1-6: unreduced samples; lanes 7-12: samples reduced byaddition of β-mercaptoethanol. Lanes 1, 2, 7, 8: BoNT/Aad^(tev); lanes3, 4, 9, 10: ΔLC-Peptide-BoNT/A^(tev); lanes 5, 6, 11, 12:ΔLC-GFP-BoNT/A^(tev). FIG. 8A: 10.5-14% Criterion gel (Bio-Rad) stainedwith Bio-Safe Coomassie (Bio-Rad); odd lanes: 0.3 μg samples; evenlanes: 1.0 μg samples. FIG. 8B: Western blot probed with polyclonalantibody Pol001 raised against BoNT/A holotoxoid (Staten Serum Institut,Denmark); odd lanes: 3 ng samples; even lanes: 10 ng samples. FIG. 8C,Western blot probed with monoclonal antibody against GFP (Clontech); oddlanes: 3 ng samples; even lanes: 10 ng samples.

FIG. 9 is an illustration of the chemical synthesis of cargo attachmentto Clostridium botulinum propeptides using the S6 peptide of SEQ ID NO:2(i.e., GDSLSWLLRLLN).

FIG. 10 is a Western blot illustrating that BoNT/A ad competes with wtBoNT/A for binding to receptors and protects SNAP 25 from cleavage. Ratspinal cord cells were exposed to the indicated concentration of BoNT/Aad in ice-cold culture medium supplemented with 56 mM KCl and 0.5 mMCaCl₂ for 15 min. wt BoNT/A in the same ice-cold culture medium was thenadded and incubation was continued on ice for an additional 15 min.Cells were then washed twice with ice-cold culture medium, fresh culturemedium was added, and cells were incubated at 37° C. for 3 hours. Cellswere harvested in SDS sample buffer, and lysates were analyzed byWestern blot using anti-SNAP 25 antibody (Synaptic Systems).

FIGS. 11A-C are images of immunostaining that show symptoms of BoNT/A adpoisoning are concurrent with BoNT/A ad LC accumulation at theneuromuscular junction in vivo. 6 week old mice were injected ip with 1μg of BoNT/A ad. The first signs of physiological effect of BoNT/A adwere observed ˜4 hours after injection (low movement activity, ruffledfur, heavy breathing), and were pronounced at the time of animaleuthanasia 12 hours after injection. Triangularis sterni nerve-musclepreparations were stained with primary antibody and probed withAlexaFluor 555-conjugated secondary antibody and Alexa-Fluor488-conjugated α-bungarotoxin. FIG. 11A illustrates the postsynapticacetylcholine receptors; FIG. 11B is the BoNT/A LC (Mab F1-40); FIG. 11Cis a merge of images shown in FIGS. 11A and B. The scale bar is 5 μm.

FIG. 12 is a series of images of immunostaining showing continuousuptake of BoNT/A ad in the primary culture of rat hippocampal neurons.Row 1 shows results after culture was incubated with 15 nM BoNT/A ad for30 mins. Row 2 shows results after culture was incubated with the sameconcentration of BoNT/A ad for 90 minutes, followed by 30 min chase withthe BoNT/A ad-free medium. Row 3 shows results after culture wasincubated with the same concentration of BoNT/A ad for 90 minutes,followed by 90 minute chase with BoNT/A ad-free medium. At the end ofincubation time, cells were washed and processed for immunocytochemicalstaining. The staining was as follows: Column A: Primary—tau mousemonoclonal IgG2b; secondary—AlexaFluor 488 conjugateddonkey-anti-IgG2b-mouse; Column B: Primary—mouse-anti-BoNT/A LCmonoclonal IgG1 (F1-40), secondary—AlexaFluor 555 conjugatedgoat-anti-IgG1-mouse; Column C: Primary rabbit-anti -SNAP 25 polyclonalantibodies, secondary—AlexaFluor 647 conjugated goatanti -rabbit IgG.Column D is a merged image of the staining from three (Row 1 and 2), ortwo (Row 3) channels. Tau from Row 3 is omitted to better visualize theco-staining pattern with internalized LC ad and SNAP 25. The scale baris equal to 10 μm.

FIGS. 13A-C are images of Western blots showing that LC ad binds SNAP 25when BoNT/A ad is internalized by rat spinal cord cells. Cultured E18rat spinal cord cells were either (1) untreated; (2) treated with 30 nMBoNT/A ad; or (3) treated with cross-linked/inactivated 30 nM BoNT/A ad.Following 8 h treatment, cells were washed and fractionated according toBernocco et al., “Sequential Detergent Fractionation of Primary Neuronsfor Proteomics Studies,” Proteomics 8 (5):930-938 (2008), which ishereby incorporated by reference in its entirety. Cytosolic extractswere immunoprecipitated with anti-SNAP 25 antibodies, and protein A andProtein G magnetic beads and separated by reduced SDS PAGE. FIG. 13Ashows results from input proteins probed with anti-BoNT/A polyclonalantibodies (Staten Serum Institut, Denmark). FIGS. 13B and C show theWestern blot of immunoprecipitates. FIG. 13B shows results from probewith mouse monoclonal anti-LC; FIG. 13C shows results from probe withrabbit anti-SNAP 25.

FIGS. 14A and B are images showing internalization of palmitoylatedΔLC-GFP-BoNT/A in COS7 cells. Palmitoylated derivative (25 nM) was addedto COS7 cells and cells were incubated for 5 minutes (FIG. 14A), or for1 hour (FIG. 14B). Image scanning was performed on a Nikon LSM 510confocal microscope equipped with argon laser, producing an excitationline of 488 nm. The scale bar is equal to 25 μm.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to an isolated Clostridiumbotulinum neurotoxin propeptide. The propeptide has a light chainregion; a heavy chain region, where the light and heavy chain regionsare linked by a disulfide bond; an intermediate region connecting thelight and heavy chain regions and comprising a highly specific proteasecleavage site which has three or more specific adjacent amino acidresidues that are recognized by the highly specific protease in order toenable cleavage; and a peptide sequence to enable site-specificattachment of cargo, where the peptide sequence is positioned upstreamof the light chain region and is separated from the N-terminus of thelight chain region by an amino acid spacer sequence.

BoNT/A propeptide has two chains, a light chain of Mr ˜50,000 and aheavy chain of Mr ˜100,000, linked by a disulfide bond between Cys₄₂₉and Cys₄₅₃. All seven BoNT serotype propeptides have a light chainregion and a heavy chain region linked by a disulfide bond. Twoessential Cys residues, one adjacent to the C-terminus of the lightchain, and a second adjacent to the N-terminus of the heavy chain arepresent in all seven BoNT serotypes. These two Cys residues form thesingle disulfide bond holding the HC and LC polypeptides together in themature neurotoxin. This disulfide bond enables the mature neurotoxin toaccomplish its native physiological activities by permitting the HC andLC to carry out their respective biological roles in concert. Theintermediate region (i.e., Lys₄₃₈-Lys₄₄₈ of BoNT/A) identifies the aminoacids eliminated during maturation of wild-type BoNT/A, and believed tobe excised by a protease endogenous to the host microorganism. Thiscleavage event generates the biologically active BoNT HC-LC dimer.

All seven BoNT serotypes contain Lys or Arg residues in the intermediateregion, which make the propeptides susceptible to activation by trypsin.Native BoNT/A propeptide recovered from young bacterial cultures can beactivated by trypsinolysis, with production of intact, S—S bound lightand heavy chain. Though multiple additional trypsin-susceptible sitesare present in the propeptides, they are resistant to proteolysis due totheir spatial positions within the native toxin molecule (Dekleva etal., “Nicking of Single Chain Clostridium botulinum Type A Neurotoxin byan Endogenous Protease,” Biochem. Biophys. Res. Commun. 162:767-772(1989); Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type Aand Implications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998),which are hereby incorporated by reference in their entirety). A secondsite in the native propeptide of several BoNT serotypes can besusceptible to trypsin cleavage when subjected to higher enzymeconcentrations or incubation times (Chaddock et al., “Expression andPurification of Catalytically Active, Non-Toxic EndopeptidaseDerivatives of Clostridium botulinum Toxin Type A,” Protein Expr. Purif.25:219-228 (2002), which is hereby incorporated by reference in itsentirety). This trypsin-susceptible site is located in the regionadjacent to the toxin receptor binding domain. This region of the HCpeptide is found to be exposed to solvent in BoNT serotypes for whichinformation is available on their 3-D crystal structure (Lacy et al.,“Crystal Structure of Botulinum Neurotoxin Type A and Implications forToxicity,” Nat. Struct. Biol. 5:898-902 (1998); Swaminathan et al.,“Structural Analysis of the Catalytic and Binding Sites of Clostridiumbotulinum Neurotoxin B,” Nat. Struct. Biol. 7:693-699 (2000), which arehereby incorporated by reference in their entirety).

Propeptides of the present invention have an intermediate regionconnecting the light and heavy chain regions which has a highly specificprotease cleavage site and no low-specificity protease cleavage sites.For purposes of the present invention, a highly specific proteasecleavage site has three or more specific adjacent amino acid residuesthat are recognized by the highly specific protease in order to permitcleavage (e.g., an enterokinase cleavage site or a TEV recognitionsequence). In contrast, a low-specificity protease cleavage site has twoor less adjacent amino acid residues that are recognized by a proteasein order to enable cleavage (e.g., a trypsin cleavage site).

In all seven BoNT serotypes, the amino acid preceding the N-terminus ofthe heavy chain is a Lys or Arg residue which is susceptible toproteolysis with trypsin. This trypsin-susceptible site can be replacedwith a five amino acid enterokinase cleavage site (i.e., DDDDK (SEQ IDNO:8)) upstream of the heavy chain's N-terminus, as illustrated by theamino acids on the dark shading in FIG. 1. Alternatively, thetrypsin-susceptible site can be replaced with a TEV recognitionsequence, as shown by the amino acid sequence on light shading inFIG. 1. Either of these modifications enables standardization activationwith specific enzymes. In serotypes A and C, additional Lys residueswithin this region may be mutated to either Gln or His, therebyeliminating additional trypsin-susceptible sites which might result inundesirable non-specific activation of the toxin. Trypsin-susceptiblerecognition sequences also occur upstream of the heavy chain'sreceptor-binding domain in serotypes A, E, and F. This region'ssusceptibility to proteolysis is consistent with its exposure to solventin the toxin's 3-D structure, as shown by X-ray crystallographyanalysis. Therefore, in serotypes A, E, and F, the susceptible residuesare modified to Asn.

Propeptides of the present invention also include a cargo attachmentpeptide sequence to enable site-specific attachment of cargo (i.e., acargo attachment peptide sequence or cargo attachment peptide). Examplesof cargo attachment peptides include an S6 sequence having a sequenceaccording to SEQ ID NO:2. The S6 sequence enables site specificattachment of cargo using Sfp phosphopantetheinyl transferase from B.subtilis, which targets the S₃ amino acid of SEQ ID NO:2 as a substrate.Zhou et al., “Genetically Encoded Short Peptide Tags for OrthogonalProtein Labeling by Sfp and AcpS Phosphopantetheinyl Transferases,” ACSChem. Biol. 2(5): 337-346 (2007), which is hereby incorporated byreference in its entirety. In addition, an N-terminally placed 12 aasequence GDSLDMLEWSLM (“A1”) (SEQ ID NO:45) enables site specificattachment of cargo using AcpS phosphopantetheinyl transferase from E.coli, which targets the S₃ amino acid of SEQ ID NO:45. Zhou et al.,“Genetically Encoded Short Peptide Tags for Orthogonal Protein Labelingby Sfp and AcpS Phosphopantetheinyl Transferases,” ACS Chem. Biol. 2(5):337-346 (2007), which is hereby incorporated by reference in itsentirety. Another example of a cargo attachment peptide sequence is theN-terminally placed 8 amino acid sequence DSLDMLEW (“A4”) (SEQ ID NO:46)that enables site specific attachment of cargo using AcpSphosphopantetheinyl transferase from E. coli, which targets the S₂ aminoacid SEQ ID NO:46 as a substrate. Zhou et al., “An Eight ResidueFragment of an Acyl Carrier Protein Suffices for Post-translationalIntroduction of Fluorescent Pantetheinyl Arms in Protein Modification invitro and in vivo,” J. Am. Chem. Soc. 130(30): 9925-9930 (2008), whichis hereby incorporated by reference in its entirety. Yet another exampleis an N-terminally placed amino acid sequence (e.g., MSGLVDIFEAQKIEWH(SEQ ID NO:47)) that enables site-specific attachment of cargo usingbiotin ligase, which targets the K₁₂ amino acid of SEQ ID NO:47 as asubstrate. The N-terminally placed 7 amino acid sequence PKPQQFM (“Qtag”) (SEQ ID NO:48) enables site-specific attachment of cargo usingtransglutaminase, which targets the QQ amino acids of SEQ ID NO:48 as asubstrate. Lin et al., “Transglutaminase-catalyzed Site-specificConjugation of Small-molecule Probes to Proteins in vitro and on theSurface of Living Cells,” J Am Chem Soc. 128 (14): 4542-4543 (2006),which is hereby incorporated by reference in its entirety. TheN-terminally placed 5 amino acid sequence GGGGG (SEQ ID NO:49) enablessite specific attachment of cargo using sortase A from S. aureus, whichtargets the N-terminus of the G₁ amino acid of SEQ ID NO:49 as asubstrate. Antos et al., “Site-specific N- and C-terminal Labeling of aSingle Polypeptide Using Sortases of Different Specificity,” J. Am.Chem. Soc. 131(31):10800-10801 (2009), which is hereby incorporated byreference in its entirety.

Propeptides of the present invention may include a signal peptidecoupled to the cargo attachment peptide sequence, suitable to permitsecreation of the neurotoxin propeptide from a eukaryotic cell to amedium. Coupling of a signal peptide sequence to the S6 peptide sequenceis illustrated in the propeptides shown in FIG. 1 by the “SP”designation.

Propeptides of the present invention may additionally include a 10-Hisaffinity tag positioned between and connecting the signal peptide to thepeptide sequence. The designation “10 His” in FIG. 1 illustrates theplacement of the polyhistidine tag.

Propeptides of the present invention may additionally include a TEVrecognition sequence positioned between and connecting the 10-Hisaffinity tag to the cargo attachment peptide. For example, as shown inFIG. 1 by the designation “TEV”, a TEV sequence is positioned upstreamof the S6 peptide sequence and downstream from the signal peptide.

Propeptides of the present invention may additionally include an 8 aminoacid StrepTag II connected to the propeptide at the C-terminus (see FIG.1, “StrepTag II”).

In one embodiment, the entire catalytic domain of the light chain regionhas been removed from the isolated propeptide. According to thisembodiment, the propeptide may have a fluorophore connected to theN-terminus of the light chain region.

Attachment of cargo to the propeptides of the present invention can befacilitated by the cargo attachment peptide sequence, an amino acidsequence that allows site-selective enzyme-specific attachment of cargoto the propeptide. For example, the chemistry involved in attachingcargo to the propeptide via the S6 peptide is illustrated in FIG. 9.Specifically, CoA derivatives, having the structure

where R (i.e., the cargo) is any prosthetic group, can be biochemicallycoupled to CoA, with the resulting CoA adduct recognized as a substratefor Sfp phosphopantetheinyl transferase used for enzymatic labeling, asdescribed in Zhou et al., “Genetically Encoded Short Peptide Tags forOrthogonal Protein Labeling by Sfp and AcpS PhosphopantetheinylTransferases,” ACS Chemical Biology 2(5):337-346 (2007), which is herebyincorporated by reference in its entirety.

Suitable cargo for attachment to propeptides of the present inventionmay include, without limitation, lipid moieties, therapeutic agents,marker molecules, and targeting agents.

Exemplary lipid moieties include fatty acids (e.g., saturated,unsaturated, greater than four carbon chain length, prostanoids,leukotienes, ecosanoids, etc.), neutral lipids (e.g., cholesterol andesters thereof, triglycerides, steroids, spermaceti (cetyl palmitate),waxes, fatty alcohols, etc.), phospholipids (e.g., phosphatidyl choline,phosphatidyl serine, ethanolamine, phosphatidyl inositol, plateletactivating factor, fatty acid glycerol ethers, cardiolipids, etc.), andcomplex lipids (e.g., sphingolipids, ceramides, glycolipids,gangliosides, sulfolipids, etc.). In one particular embodiment, thelipid is selected from a group consisting of palmitoyl-CoA, C-22aliphatic CoA, or cholesterol CoA. Incorporation of lipid moieties intothe propeptide of the present invention anchors the propeptides toplasma membranes at the injection site and restricts its diffusion awayfrom the site of intended action.

Exemplary therapeutic agents may include, without limitation, thepeptide described in Zuniga et al., “A Potent Peptidomimetic Inhibitorof Botulinum Neurotoxin Serotype A Has a Very Different Conformationthan SNAP-25 Substrate,” Structure 16:1588-1597 (2008) (which is herebyincorporated by reference in its entirety), which is an effective BoNTinhibitor. Other therapeutic agents include any agent with a therapeutictarget in, e.g., the neural cytosol including, without limitation,agents for treating neuropathic pain, Alzheimer's Disease, and virusinhibitors (e.g., HSV2 inhibitors).

Exemplary marker molecules include, without limitation, fluorophoreshaving a photoluminescent property that can be detected and easilyidentified with appropriate detection equipment to permit neurotoxintrafficking studies. Exemplary fluorescent labels include, withoutlimitation, fluorescent dyes, semiconductor quantum dots, lanthanideatom-containing complexes, and fluorescent proteins. The fluorophoreused in the present invention is characterized by a fluorescent emissionmaxima that is detectable either visually or using optical detectors ofthe type known in the art.

Exemplary dyes include, without limitation, Cy2™, YO-PRO™-1, YOYO™-1,Calcein, FITC, FluorX™, Alexa™, Rhodamine 110, 5-FAM, Oregon Green™ 500,Oregon Green™ 488, RiboGreen™, Rhodamine Green™, Rhodamine 123,Magnesium Green™, Calcium Green™, TO-PRO™-1, TOTO®-1, JOE, BODIPY®530/550, Dil, BODIPY® TMR, BODIPY® 558/568, BODIPY® 564/570, Cy3™,Alexa™ 546, TRITC, Magnesium Orange™, Phycoerythrin R&B, RhodaminePhalloidin, Calcium Orange™, Pyronin Y, Rhodamine B, TAMRA, RhodamineRed™, Cy3.5™, ROX, Calcium Crimson™, Alexa™ 594, TEXAS RED®, Nile Red,YO-PRO™-3, YOYO™-3, R-phycocyanin, C-Phycocyanin, TO -PRO™-3, TOTO®-3,DiD DilC(5), CyS™, Thiadicarbocyanine, and Cy5.5™. Other dyes now knownor hereafter developed can similarly be used as long as their excitationand emission characteristics are compatible with a light source andnon-interfering with other fluorophores that may be present.

Exemplary proteins include, without limitation, both naturally occurringand modified (i.e., mutant) green fluorescent proteins (Prasher et al.,Gene 111:229-233 (1992); PCT Application WO 95/07463, which are herebyincorporated by reference in their entirety) from various sources suchas Aequorea and Renilla; both naturally occurring and modified bluefluorescent proteins (Karatani et al., Photochem. Photobiol.55(2):293-299 (1992); Lee et al., Methods Enzymol. (Biolumin.Chemilumin.) 57:226-234 (1978); Gast et al., Biochem. Biophys. Res.Commun. 80(1):14-21 (1978), which are hereby incorporated by referencein their entirety) from various sources such as Vibrio andPhotobacterium; and phycobiliproteins of the type derived fromcyanobacteria and eukaryotic algae (Apt et al., J. Mol. Biol. 238:79-96(1995); Glazer, Ann. Rev. Microbiol. 36:173-198 (1982); Fairchild etal., J. Biol. Chem. 269:8686-8694 (1994); Pilot et al., Proc. Natl.Acad. Sci. USA 81:6983-6987 (1984); Lui et al., Plant Physiol.103:293-294 (1993); Houmard et al., J. Bacteriol. 170:5512-5521 (1988),which are hereby incorporated by reference in their entirety), severalof which are commercially available from ProZyme, Inc. (San Leandro,Calif.). Other fluorescent proteins now known or hereafter developed cansimilarly be used as long as their excitation and emissioncharacteristics are compatible with the light source and non-interferingwith other fluorophores that may be present.

Exemplary lanthanide atoms include, without limitation, Ce, Pr, Nd, Pm,Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lv. Of these, Nd, Er, and Tb arepreferred because they are commonly used in imaging applications.

Exemplary targeting agents may include, without limitation, agents thatdirect trafficking of Botulinum neurotoxins to specific neurons andother cell types. Ligands for cell receptors (e.g., NGF, EGF, andothers) and antibodies against receptors may also be used as targetingagents. Selectively incorporating lipid moieties into recombinant BoNT/Aderivatives by enzymatic coupling to the S6 peptide tag, in order torestrict the diffusion of the protein adduct from the site of injection,is described herein in the Examples.

Propeptides of the present invention may include a disabling mutation inan active metalloprotease site of the propeptide.

As noted supra, propeptides of the present invention may have a cargoattachment peptide sequence to enable site-specific attachment of cargo.The cargo attachment peptide is positioned upstream of the light chainregion and is separated from the N-terminus of the light chain region byan amino acid spacer sequence. The amino acid spacer (or linker)sequence may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-35, or 36-40, or more,amino acid residues. The amino acid spacer (or linker) sequence servesto preserve and protect conformational independence of the cargoattachment peptide and the botulinum neurotoxin. An exemplary amino acidspacer (or linker) sequence is shown in FIG. 1 (bold type) as the 7amino acid spacer ARGGASG (SEQ ID NO:9).

Propeptides of the present invention may further include aneuron-specific protease cleavage site. In one embodiment, the cargo isattached at the N-terminus of the propeptide's light chain via a peptidelinker carrying a neuron-specific protease cleavage site (e.g., a BACE1cleavage site). In one embodiment, the neuron-specific cleavage site maybe positioned between the cargo attachment peptide and the linker orspacer sequence. In one embodiment in which the cargo is a lipid moiety,the lipidated propeptide will remain anchored to the membrane until itis released by endogenous neuron-specific protease and will beinternalized by interaction with endogenous neuronal receptors, leadingto release of the active light chain into the neuronal cytoplasm. Innon-neuronal cells, which are neuron-specific protease deficient,lipidated propeptide can only be internalized through a non-specificmechanism and directed into a degradation pathway, thereby contributingto removal of excess propeptide from the circulation.

Propeptides of the present invention may also possess a non-native motifin the light chain region that is capable of inactivating light chainmetalloprotease activity in a toxic Botulinum neurotoxin, as describedin U.S. Patent Application Publication No. 2006/0204524 to Ichtchenko etal., which is hereby incorporated by reference in its entirety.

In one embodiment, propeptides of the present invention have light andheavy chains that are not truncated. In another embodiment, the entirecatalytic domain of the light chain has been removed.

Another aspect of the present invention relates to isolated nucleic acidmolecules encoding propeptides of the present invention.

Wildtype BoNT/A has an amino acid sequence as set forth in GenBankAccession No. ABP48106 (SEQ ID NO:7), as follows:

MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN 60PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS TDLGRMLLTS IVRGIPFWGG 120STIDTELKVI DTNCINVIQP DGSYRSEELN LVIIGPSADI IQFECKSFGH EVLNLTRNGY 180GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GKFATDPAVT LAHELIHAGH RLYGIAINPN 240RVFKVNTNAY YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA 300KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT EDNFVKFFKV 360LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN FNGQNTEINN MNFTKLKNFT 420GLFEFYKLLC VRGIITSKTK SLDKGYNKAL NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE 480ITSDTNIEAA EENISLDLIQ QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG 540KKYELDKYTM FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA 600AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD DFVGALIFSG 660AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS KRNEKWDEVY KYIVTNWLAK 720VNTQIDLIRK KMKEALENQA EATKAIINYQ YNQYTEEEKN NINFNIDDLS SKLNESINKA 780MININKFLNQ CSVSYLMNSM IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK 840VNNTLSTDIP FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI 900GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP KYFNSISLNN 960EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK YSQMINISDY INRWIFVTIT 1020NNRLNNSKIY INGRLIDQKP ISNLGNIHAS NNIMFKLDGC RDTHRYIWIK YFNLFDKELN 1080EKEIKDLYDN QSNSGILKDF WGDYLQYDKP YYMLNLYDPN KYVDVNNVGI RGYMYLKGPR 1140GSVMTTNIYL NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA 1200GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI GFHQFNNIAK 1260LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPL

An exemplary nucleic acid molecule of the present invention is set forthin GenBank Accession No. GQ855201 (SEQ ID NO:15), as follows:

gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 60gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 120acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 180agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 240ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 300ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 360taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 420aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat 480gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 540agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 600catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 660ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 720atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 780ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 840gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 900ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 960ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1020gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1080ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1140gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1200ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1260gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1320gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 1380caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 1440cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 1500ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 1560taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 1620tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 1680gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 1740agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 1800aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 1860gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 1920gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 1980tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 2040agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2100cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2160gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2220gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 2280ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 2340cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 2400cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct 2460ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga 2520caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag 2580acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt 2640tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga 2700ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac 2760aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg 2820tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg 2880ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca 2940tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact 3000gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc 3060gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta 3120cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 3180ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg 3240agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 3300ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca 3360tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 3420acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa 3480ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca 3540ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 3600cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc 3660ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg 3720cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt 3780ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt 3840gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa 3900tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt 3960ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca 4020ccatcgggcg cggatcccgg tccgttcgaa ccagaactct ggaagcttaa ctcctaaaaa 4080accgccacca tgaaattctt agtcaacgtt gcccttgttt ttatggtcgt atacatttct 4140tacatctatg cggccgctca tcaccaccat catcaccatc accaccacac gcgtgaaaac 4200ctgtattttc agggcgccgg tgactccctg tcttggctgc tccgtctgct caacgcgcgc 4260ggtggagcta gcggcccgtt cgttaacaaa caatttaact acaaggatcc tgtcaatggt 4320gtggacattg cctatattaa gatcccgaat gcgggtcaga tgcaacccgt gaaagcattc 4380aagatccaca acaaaatctg ggtcatccct gaacgtgaca ctttcacaaa ccctgaagag 4440ggcgacctca accctccccc agaagccaaa caggttccgg tgtcttacta cgatagcacg 4500tacttgtcca ccgataacga gaaggacaac tacctgaagg gagtgaccaa gttgtttgag 4560aggatctact ctaccgatct cggacgtatg ctgctcacga gcattgtgcg cggtatccca 4620ttctggggcg gttcaaccat tgatacagaa ctgaaagtca ttgacactaa ttgtatcaac 4680gttattcaac cagatggcag ctaccgttcc gaggaattga acttggtcat cattggtcca 4740tccgcagaca tcattcagtt tgaatgcaaa tccttcggtc acgaagtgct caacctgacg 4800cgcaacggtt acggctccac ccagtacatc cgtttcagcc ctgatttcac atttggcttc 4860gaggaaagcc tggaggttga caccaacccg ctcctgggtg ctggcaagtt tgcaaccgat 4920cccgcggtga ctctcgctca tgctctgatc cacgccggac accgcctcta tggcatcgct 4980atcaatccga accgcgtgtt caaagtgaat acgaacgcct actatgagat gagcggtctg 5040gaggtttcct ttgaggaact gagaaccttc ggcggtcacg atgccaagtt catcgacagc 5100ttgcaggaaa atgagtttcg cctgtactat tacaacaagt ttaaagacat cgcttccaca 5160ttgaacaaag ccaagtcaat cgtgggtacg acagcttcat tgcagtatat gaagaatgtt 5220ttcaaggaga aatacttgct gtcagaggat acctctggca agttctctgt ggacaaactg 5280aaattcgaca aactgtacaa gatgctgacc gagatttata cggaagataa ctttgtgaaa 5340ttcttcaaag tcctcaacag gaaaactgct ctgaactttg acaaggctgt gttcaagatc 5400aacatcgtcc ccaaagttaa ctacacaatc tatgatggat tcaatctgag aaacaccaac 5460ttggccgcca acttcaacgg ccaaaatacc gaaattaata acatgaattt caccaaactg 5520aagaacttta ctggactgtt cgagttctac aagctgctct gcgtgcgtgg catcatcacc 5580tcacatactc agtctctaga ccagggttat aacgacgatg acgataaagc tctgaacgat 5640ctgtgtatca aggtgaataa ctgggatctg ttctttagcc caagcgagga taacttcacg 5700aacgatctca acaaaggtga agagatcacg tctgatacca atatcgaagc ggctgaagag 5760aatatctcct tggatctcat ccagcaatat tacctgacct ttaacttcga taacgagccc 5820gaaaacatct ccatcgagaa cctcagctca gacatcattg gtcagttgga gctgatgcca 5880aacattgaac gcttccccaa cggcaagaaa tacgaactcg acaagtatac gatgtttcat 5940tacttaagag cgcaggagtt tgaacacggc aagagccgca ttgctctcac taactccgtg 6000aatgaagccc tgctcaatcc gtcaagggtg tacacattct ttagctccga ctatgtcaag 6060aaagtgaaca aagccaccga agcggcaatg ttcctgggat gggttgaaca actggtctac 6120gacttcaccg acgagacctc tgaggtgagc acaacggaca agattgctga catcactatc 6180attatcccgt atattggacc tgccttgaat attggcaaca tgctctacaa agacgatttc 6240gttggtgccc tgatcttcag cggtgccgtg atcctgttgg agttcattcc tgaaatcgcc 6300atccctgtgc tgggcacgtt cgctctggtc tcatacattg cgaataaggt cttgaccgtg 6360cagacaatcg ataatgccct ctccaaacgt aacgaaaaat gggacgaggt ctacaaatac 6420atcgtgacca actggctggc aaaggttaac acccaaattg atctgatccg taagaaaatg 6480aaggaggctt tggagaacca ggctgaagct actaaagcca ttatcaacta ccagtataat 6540cagtatacag aagaggaaaa gaataacatc aatttcaaca tcgatgactt gtcctcaaag 6600ctgaacgagt ccatcaacaa agctatgatc aacatcaaca aattcctgaa tcagtgctcc 6660gtgtcttacc tgatgaactc tatgatccca tacggtgtga agcgcctgga ggacttcgat 6720gccagcctga aagacgcact gctcaaatac atttacgata atcgcggcac tttgattggc 6780caagttgacc gtctgaagga caaggttaac aataccttgt caaccgatat cccctttcaa 6840ctgtccaaat acgttgataa ccagcgcttg ctctctactt tcaccgaata cattaacaac 6900attatcaata catcaattct caacctgcgc tatgagtcca atcatctgat cgatctgtct 6960cgttacgcca gcaagatcaa cattggcagc aaagtgaact tcgatccgat tgacaagaac 7020caaatccagt tgttcaacct cgaaagctcc aaaatcgaag tgatcctgaa gaatgccatc 7080gtctacaact ccatgtatga aaatttctca acttcattct ggattagaat cccgaaatac 7140ttcaactcaa tctctctgaa taacgaatac acgatcatta actgtatgga gaataactct 7200ggttggaagg tttccttgaa ctatggagaa attatctgga ctctgcaaga tacgcaagag 7260atcaaacagc gtgtggtctt taaatacagc cagatgatta acatctctga ctacatcaac 7320agatggatct ttgtcaccat tacaaacaat cgcctgaata actccaaaat ctacatcaac 7380ggtcgtctga tcgaccagaa acctatttca aacctcggca acattcatgc ttccaataac 7440atcatgttta agttggatgg ttgccgcgat acccaccgtt acatctggat caagtatttc 7500aatctgttcg acaaagaact caatgagaaa gagatcaaag acttgtatga taatcagtca 7560aactccggca ttctgaaaga cttctggggc gattacctcc agtacgataa gccatattac 7620atgctgaatc tctatgaccc taacaaatat gtggacgtga acaatgtcgg tatccgtggc 7680tacatgtacc tcaaaggacc acgtggtagc gttatgacaa ccaacatcta cctgaatagc 7740tccttgtatc gcggtacgaa gttcattatc aagaagtacg cttcaggcaa caaggacaac 7800atcgtgagga acaatgatcg cgtgtacatc aacgtcgtgg tgaagaataa ggaataccgc 7860ttggcgacca acgcttctca ggctggagtt gagaagatcc tgagcgcctt ggagatccca 7920gacgttggca acctgagcca agtggttgtg atgaaaagca agaatgacca gggaatcacc 7980aacaaatgca aaatgaacct gcaagacaac aacggcaatg acatcggttt catcggtttc 8040caccagttta acaatattgc gaagctggtc gccagcaact ggtacaacag gcagattgag 8100aggtcatccc gtaccttagg atgctcttgg gaatttatcc ccgtggacga tggttggggc 8160gagagacccc tgggcgcagg ttggtcccac cctcagttcg agaagtaata gttaatagat 8220aataatagct cgaggcatgc gagctccctc aggaggccta cgtcgacgag ctcactagtc 8280gcggccgctt tcgaatctag agcctgcagt ctcgaggcat gcggtaccaa gcttgtcgag 8340aagtactaga ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa 8400aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac 8460ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 8520aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 8580catgtctgga tctgatcact gcttgagcct aggagatccg aaccagataa gtgaaatcta 8640gttccaaact attttgtcat ttttaatttt cgtattagct tacgacgcta cacccagttc 8700ccatctattt tgtcactctt ccctaaataa tccttaaaaa ctccatttcc acccctccca 8760gttcccaact attttgtccg cccacagcgg ggcatttttc ttcctgttat gtttttaatc 8820aaacatcctg ccaactccat gtgacaaacc gtcatcttcg gctacttttt ctctgtcaca 8880gaatgaaaat ttttctgtca tctcttcgtt attaatgttt gtaattgact gaatatcaac 8940gcttatttgc agcctgaatg gcgaatggThis nucleotide sequence encodes the neurotoxin BoNT/Aad^(ek).

Another exemplary nucleic acid molecule of the present invention is setforth in GenBank Accession No. GQ855202 (SEQ ID NO:10), as follows:

gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 60gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 120acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 180agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 240ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 300ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 360taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 420aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat 480gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 540agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 600catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 660ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 720atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 780ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 840gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 900ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 960ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1020gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1080ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1140gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1200ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1260gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1320gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 1380caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 1440cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 1500ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 1560taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 1620tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 1680gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 1740agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 1800aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 1860gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 1920gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 1980tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 2040agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2100cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2160gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2220gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 2280ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 2340cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 2400cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct 2460ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga 2520caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag 2580acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt 2640tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga 2700ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac 2760aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg 2820tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg 2880ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca 2940tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact 3000gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc 3060gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta 3120cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 3180ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg 3240agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 3300ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca 3360tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 3420acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa 3480ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca 3540ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 3600cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc 3660ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg 3720cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt 3780ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt 3840gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa 3900tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt 3960ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca 4020ccatcgggcg cggatcccgg tccgttcgaa ccagaactct ggaagcttaa ctcctaaaaa 4080accgccacca tgaaattctt agtcaacgtt gcccttgttt ttatggtcgt atacatttct 4140tacatctatg cggccgctca tcaccaccat catcaccatc accaccacac gcgtgaaaac 4200ctgtattttc agggcgccgg tgactccctg tcttggctgc tccgtctgct caacgcgcgc 4260ggtggagcta gcggcccgtt cgttaacaaa caatttaact acaaggatcc tgtcaatggt 4320gtggacattg cctatattaa gatcccgaat gcgggtcaga tgcaacccgt gaaagcattc 4380aagatccaca acaaaatctg ggtcatccct gaacgtgaca ctttcacaaa ccctgaagag 4440ggcgacctca accctccccc agaagccaaa caggttccgg tgtcttacta cgatagcacg 4500tacttgtcca ccgataacga gaaggacaac tacctgaagg gagtgaccaa gttgtttgag 4560aggatctact ctaccgatct cggacgtatg ctgctcacga gcattgtgcg cggtatccca 4620ttctggggcg gttcaaccat tgatacagaa ctgaaagtca ttgacactaa ttgtatcaac 4680gttattcaac cagatggcag ctaccgttcc gaggaattga acttggtcat cattggtcca 4740tccgcagaca tcattcagtt tgaatgcaaa tccttcggtc acgaagtgct caacctgacg 4800cgcaacggtt acggctccac ccagtacatc cgtttcagcc ctgatttcac atttggcttc 4860gaggaaagcc tggaggttga caccaacccg ctcctgggtg ctggcaagtt tgcaaccgat 4920cccgcggtga ctctcgctca tgctctgatc cacgccggac accgcctcta tggcatcgct 4980atcaatccga accgcgtgtt caaagtgaat acgaacgcct actatgagat gagcggtctg 5040gaggtttcct ttgaggaact gagaaccttc ggcggtcacg atgccaagtt catcgacagc 5100ttgcaggaaa atgagtttcg cctgtactat tacaacaagt ttaaagacat cgcttccaca 5160ttgaacaaag ccaagtcaat cgtgggtacg acagcttcat tgcagtatat gaagaatgtt 5220ttcaaggaga aatacttgct gtcagaggat acctctggca agttctctgt ggacaaactg 5280aaattcgaca aactgtacaa gatgctgacc gagatttata cggaagataa ctttgtgaaa 5340ttcttcaaag tcctcaacag gaaaactgct ctgaactttg acaaggctgt gttcaagatc 5400aacatcgtcc ccaaagttaa ctacacaatc tatgatggat tcaatctgag aaacaccaac 5460ttggccgcca acttcaacgg ccaaaatacc gaaattaata acatgaattt caccaaactg 5520aagaacttta ctggactgtt cgagttctac aagctgctct gcgtgcgtgg catcatcacc 5580tcacatactc agtctctaga ccagggtggc gagaacctgt acttccaggg tgctctgaac 5640gatctgtgta tcaaggtgaa taactgggat ctgttcttta gcccaagcga ggataacttc 5700acgaacgatc tcaacaaagg tgaagagatc acgtctgata ccaatatcga agcggctgaa 5760gagaatatct ccttggatct catccagcaa tattacctga cctttaactt cgataacgag 5820cccgaaaaca tctccatcga gaacctcagc tcagacatca ttggtcagtt ggagctgatg 5880ccaaacattg aacgcttccc caacggcaag aaatacgaac tcgacaagta tacgatgttt 5940cattacttaa gagcgcagga gtttgaacac ggcaagagcc gcattgctct cactaactcc 6000gtgaatgaag ccctgctcaa tccgtcaagg gtgtacacat tctttagctc cgactatgtc 6060aagaaagtga acaaagccac cgaagcggca atgttcctgg gatgggttga acaactggtc 6120tacgacttca ccgacgagac ctctgaggtg agcacaacgg acaagattgc tgacatcact 6180atcattatcc cgtatattgg acctgccttg aatattggca acatgctcta caaagacgat 6240ttcgttggtg ccctgatctt cagcggtgcc gtgatcctgt tggagttcat tcctgaaatc 6300gccatccctg tgctgggcac gttcgctctg gtctcataca ttgcgaataa ggtcttgacc 6360gtgcagacaa tcgataatgc cctctccaaa cgtaacgaaa aatgggacga ggtctacaaa 6420tacatcgtga ccaactggct ggcaaaggtt aacacccaaa ttgatctgat ccgtaagaaa 6480atgaaggagg ctttggagaa ccaggctgaa gctactaaag ccattatcaa ctaccagtat 6540aatcagtata cagaagagga aaagaataac atcaatttca acatcgatga cttgtcctca 6600aagctgaacg agtccatcaa caaagctatg atcaacatca acaaattcct gaatcagtgc 6660tccgtgtctt acctgatgaa ctctatgatc ccatacggtg tgaagcgcct ggaggacttc 6720gatgccagcc tgaaagacgc actgctcaaa tacatttacg ataatcgcgg cactttgatt 6780ggccaagttg accgtctgaa ggacaaggtt aacaatacct tgtcaaccga tatccccttt 6840caactgtcca aatacgttga taaccagcgc ttgctctcta ctttcaccga atacattaac 6900aacattatca atacatcaat tctcaacctg cgctatgagt ccaatcatct gatcgatctg 6960tctcgttacg ccagcaagat caacattggc agcaaagtga acttcgatcc gattgacaag 7020aaccaaatcc agttgttcaa cctcgaaagc tccaaaatcg aagtgatcct gaagaatgcc 7080atcgtctaca actccatgta tgaaaatttc tcaacttcat tctggattag aatcccgaaa 7140tacttcaact caatctctct gaataacgaa tacacgatca ttaactgtat ggagaataac 7200tctggttgga aggtttcctt gaactatgga gaaattatct ggactctgca agatacgcaa 7260gagatcaaac agcgtgtggt ctttaaatac agccagatga ttaacatctc tgactacatc 7320aacagatgga tctttgtcac cattacaaac aatcgcctga ataactccaa aatctacatc 7380aacggtcgtc tgatcgacca gaaacctatt tcaaacctcg gcaacattca tgcttccaat 7440aacatcatgt ttaagttgga tggttgccgc gatacccacc gttacatctg gatcaagtat 7500ttcaatctgt tcgacaaaga actcaatgag aaagagatca aagacttgta tgataatcag 7560tcaaactccg gcattctgaa agacttctgg ggcgattacc tccagtacga taagccatat 7620tacatgctga atctctatga ccctaacaaa tatgtggacg tgaacaatgt cggtatccgt 7680ggctacatgt acctcaaagg accacgtggt agcgttatga caaccaacat ctacctgaat 7740agctccttgt atcgcggtac gaagttcatt atcaagaagt acgcttcagg caacaaggac 7800aacatcgtga ggaacaatga tcgcgtgtac atcaacgtcg tggtgaagaa taaggaatac 7860cgcttggcga ccaacgcttc tcaggctgga gttgagaaga tcctgagcgc cttggagatc 7920ccagacgttg gcaacctgag ccaagtggtt gtgatgaaaa gcaagaatga ccagggaatc 7980accaacaaat gcaaaatgaa cctgcaagac aacaacggca atgacatcgg tttcatcggt 8040ttccaccagt ttaacaatat tgcgaagctg gtcgccagca actggtacaa caggcagatt 8100gagaggtcat cccgtacctt aggatgctct tgggaattta tccccgtgga cgatggttgg 8160ggcgagagac ccctgggcgc aggttggtcc caccctcagt tcgagaagta atagttaata 8220gataataata gctcgaggca tgcgagctcc ctcaggaggc ctacgtcgac gagctcacta 8280gtcgcggccg ctttcgaatc tagagcctgc agtctcgagg catgcggtac caagcttgtc 8340gagaagtact agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta 8400aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 8460aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 8520aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 8580tatcatgtct ggatctgatc actgcttgag cctaggagat ccgaaccaga taagtgaaat 8640ctagttccaa actattttgt catttttaat tttcgtatta gcttacgacg ctacacccag 8700ttcccatcta ttttgtcact cttccctaaa taatccttaa aaactccatt tccacccctc 8760ccagttccca actattttgt ccgcccacag cggggcattt ttcttcctgt tatgttttta 8820atcaaacatc ctgccaactc catgtgacaa accgtcatct tcggctactt tttctctgtc 8880acagaatgaa aatttttctg tcatctcttc gttattaatg tttgtaattg actgaatatc 8940aacgcttatt tgcagcctga atggcgaatg gThis nucleotide sequence encodes the neurotoxin BoNT/Aad^(tev).

Another exemplary nucleic acid molecule of the present invention is setforth in GenBank Accession No. GQ855203 (SEQ ID NO:11), as follows:

gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 60gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 120acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 180agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 240ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 300ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 360taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 420aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat 480gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 540agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 600catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 660ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 720atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 780ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 840gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 900ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 960ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1020gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1080ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1140gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1200ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1260gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1320gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 1380caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 1440cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 1500ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 1560taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 1620tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 1680gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 1740agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 1800aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 1860gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 1920gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 1980tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 2040agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2100cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2160gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2220gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 2280ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 2340cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 2400cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct 2460ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga 2520caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag 2580acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt 2640tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga 2700ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac 2760aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg 2820tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg 2880ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca 2940tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact 3000gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc 3060gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta 3120cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 3180ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg 3240agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 3300ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca 3360tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 3420acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa 3480ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca 3540ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 3600cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc 3660ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg 3720cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt 3780ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt 3840gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa 3900tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt 3960ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca 4020ccatcgggcg cggatcccgg tccgttcgaa ccagaactct ggaagcttaa ctcctaaaaa 4080accgccacca tgaaattctt agtcaacgtt gcccttgttt ttatggtcgt atacatttct 4140tacatctatg cggccgctca tcaccaccat catcaccatc accaccacac gcgtgaaaac 4200ctgtattttc agggcgccgg tgactccctg tcttggctgc tccgtctgct caacgcgcgc 4260ggtggagcta gcggcggtac caatggcaac ggtaacggtg gtaatctgag aaacaccaac 4320ttggccgcca acttcaacgg ccaaaatacc gaaattaata acatgaattt caccaaactg 4380aagaacttta ctggactgtt cgagttctac aagctgctct gcgtgcgtgg catcatcacc 4440tcacatactc agtctctaga ccagggtggc gagaacctgt acttccaggg tgctctgaac 4500gatctgtgta tcaaggtgaa taactgggat ctgttcttta gcccaagcga ggataacttc 4560acgaacgatc tcaacaaagg tgaagagatc acgtctgata ccaatatcga agcggctgaa 4620gagaatatct ccttggatct catccagcaa tattacctga cctttaactt cgataacgag 4680cccgaaaaca tctccatcga gaacctcagc tcagacatca ttggtcagtt ggagctgatg 4740ccaaacattg aacgcttccc caacggcaag aaatacgaac tcgacaagta tacgatgttt 4800cattacttaa gagcgcagga gtttgaacac ggcaagagcc gcattgctct cactaactcc 4860gtgaatgaag ccctgctcaa tccgtcaagg gtgtacacat tctttagctc cgactatgtc 4920aagaaagtga acaaagccac cgaagcggca atgttcctgg gatgggttga acaactggtc 4980tacgacttca ccgacgagac ctctgaggtg agcacaacgg acaagattgc tgacatcact 5040atcattatcc cgtatattgg acctgccttg aatattggca acatgctcta caaagacgat 5100ttcgttggtg ccctgatctt cagcggtgcc gtgatcctgt tggagttcat tcctgaaatc 5160gccatccctg tgctgggcac gttcgctctg gtctcataca ttgcgaataa ggtcttgacc 5220gtgcagacaa tcgataatgc cctctccaaa cgtaacgaaa aatgggacga ggtctacaaa 5280tacatcgtga ccaactggct ggcaaaggtt aacacccaaa ttgatctgat ccgtaagaaa 5340atgaaggagg ctttggagaa ccaggctgaa gctactaaag ccattatcaa ctaccagtat 5400aatcagtata cagaagagga aaagaataac atcaatttca acatcgatga cttgtcctca 5460aagctgaacg agtccatcaa caaagctatg atcaacatca acaaattcct gaatcagtgc 5520tccgtgtctt acctgatgaa ctctatgatc ccatacggtg tgaagcgcct ggaggacttc 5580gatgccagcc tgaaagacgc actgctcaaa tacatttacg ataatcgcgg cactttgatt 5640ggccaagttg accgtctgaa ggacaaggtt aacaatacct tgtcaaccga tatccccttt 5700caactgtcca aatacgttga taaccagcgc ttgctctcta ctttcaccga atacattaac 5760aacattatca atacatcaat tctcaacctg cgctatgagt ccaatcatct gatcgatctg 5820tctcgttacg ccagcaagat caacattggc agcaaagtga acttcgatcc gattgacaag 5880aaccaaatcc agttgttcaa cctcgaaagc tccaaaatcg aagtgatcct gaagaatgcc 5940atcgtctaca actccatgta tgaaaatttc tcaacttcat tctggattag aatcccgaaa 6000tacttcaact caatctctct gaataacgaa tacacgatca ttaactgtat ggagaataac 6060tctggttgga aggtttcctt gaactatgga gaaattatct ggactctgca agatacgcaa 6120gagatcaaac agcgtgtggt ctttaaatac agccagatga ttaacatctc tgactacatc 6180aacagatgga tctttgtcac cattacaaac aatcgcctga ataactccaa aatctacatc 6240aacggtcgtc tgatcgacca gaaacctatt tcaaacctcg gcaacattca tgcttccaat 6300aacatcatgt ttaagttgga tggttgccgc gatacccacc gttacatctg gatcaagtat 6360ttcaatctgt tcgacaaaga actcaatgag aaagagatca aagacttgta tgataatcag 6420tcaaactccg gcattctgaa agacttctgg ggcgattacc tccagtacga taagccatat 6480tacatgctga atctctatga ccctaacaaa tatgtggacg tgaacaatgt cggtatccgt 6540ggctacatgt acctcaaagg accacgtggt agcgttatga caaccaacat ctacctgaat 6600agctccttgt atcgcggtac gaagttcatt atcaagaagt acgcttcagg caacaaggac 6660aacatcgtga ggaacaatga tcgcgtgtac atcaacgtcg tggtgaagaa taaggaatac 6720cgcttggcga ccaacgcttc tcaggctgga gttgagaaga tcctgagcgc cttggagatc 6780ccagacgttg gcaacctgag ccaagtggtt gtgatgaaaa gcaagaatga ccagggaatc 6840accaacaaat gcaaaatgaa cctgcaagac aacaacggca atgacatcgg tttcatcggt 6900ttccaccagt ttaacaatat tgcgaagctg gtcgccagca actggtacaa caggcagatt 6960gagaggtcat cccgtacctt aggatgctct tgggaattta tccccgtgga cgatggttgg 7020ggcgagagac ccctgggcgc aggttggtcc caccctcagt tcgagaagta atagttaata 7080gataataata gctcgaggca tgcgagctcc ctcaggaggc ctacgtcgac gagctcacta 7140gtcgcggccg ctttcgaatc tagagcctgc agtctcgagg catgcggtac caagcttgtc 7200gagaagtact agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta 7260aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 7320aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 7380aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 7440tatcatgtct ggatctgatc actgcttgag cctaggagat ccgaaccaga taagtgaaat 7500ctagttccaa actattttgt catttttaat tttcgtatta gcttacgacg ctacacccag 7560ttcccatcta ttttgtcact cttccctaaa taatccttaa aaactccatt tccacccctc 7620ccagttccca actattttgt ccgcccacag cggggcattt ttcttcctgt tatgttttta 7680atcaaacatc ctgccaactc catgtgacaa accgtcatct tcggctactt tttctctgtc 7740acagaatgaa aatttttctg tcatctcttc gttattaatg tttgtaattg actgaatatc 7800aacgcttatt tgcagcctga atggcgaatg gThis nucleotide sequence encodes the neurotoxinΔLC-Peptide-BoNT/A^(tev).

Another exemplary nucleic acid molecule of the present invention is setforth in GenBank Accession No. GQ855204 (SEQ ID NO:12), as follows:

gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 60gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 120acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 180agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 240ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 300ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 360taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 420aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat 480gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 540agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 600catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 660ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 720atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 780ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 840gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 900ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 960ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1020gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1080ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1140gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1200ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1260gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1320gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 1380caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 1440cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 1500ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 1560taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 1620tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 1680gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 1740agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 1800aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 1860gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 1920gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 1980tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 2040agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2100cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2160gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2220gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 2280ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 2340cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 2400cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct 2460ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga 2520caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag 2580acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt 2640tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga 2700ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac 2760aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg 2820tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg 2880ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca 2940tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact 3000gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc 3060gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta 3120cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 3180ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg 3240agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 3300ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca 3360tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 3420acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa 3480ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca 3540ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 3600cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc 3660ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg 3720cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt 3780ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt 3840gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa 3900tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt 3960ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca 4020ccatcgggcg cggatcccgg tccgttcgaa ccagaactct ggaagcttaa ctcctaaaaa 4080accgccacca tgaaattctt agtcaacgtt gcccttgttt ttatggtcgt atacatttct 4140tacatctatg cggccgctca tcaccaccat catcaccatc accaccacac gcgtgaaaac 4200ctgtattttc agggcgccgg tgactccctg tcttggctgc tccgtctgct caacgcgcgc 4260ggtggagcta gcgtgagcaa gggcgccgag ctgttcaccg gcatcgtgcc catcctgatc 4320gagctgaatg gcgatgtgaa tggccacaag ttcagcgtga gcggcgaggg cgagggcgat 4380gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcctgtgccc 4440tggcccaccc tggtgaccac cctgagctac ggcgtgcagt gcttctcacg ctaccccgat 4500cacatgaagc agcacgactt cttcaagagc gccatgcctg agggctacat ccaggagcgc 4560accatcttct tcgaggatga cggcaactac aagtcgcgcg ccgaggtgaa gttcgagggc 4620gataccctgg tgaatcgcat cgagctgacc ggcaccgatt tcaaggagga tggcaacatc 4680ctgggcaata agatggagta caactacaac gcccacaatg tgtacatcat gaccgacaag 4740gccaagaatg gcatcaaggt gaacttcaag atccgccaca acatcgagga tggcagcgtg 4800cagctggccg accactacca gcagaatacc cccatcggcg atggccctgt gctgctgccc 4860gataaccact acctgtccac ccagagcgcc ctgtccaagg accccaacga gaagcgcgat 4920cacatgatct acttcggctt cgtgaccgcc gccgccatca cccacggcat ggatgagctg 4980tacaagggta ccaatggcaa cggtaacggt ggtaatctga gaaacaccaa cttggccgcc 5040aacttcaacg gccaaaatac cgaaattaat aacatgaatt tcaccaaact gaagaacttt 5100actggactgt tcgagttcta caagctgctc tgcgtgcgtg gcatcatcac ctcacatact 5160cagtctctag accagggtgg cgagaacctg tacttccagg gtgctctgaa cgatctgtgt 5220atcaaggtga ataactggga tctgttcttt agcccaagcg aggataactt cacgaacgat 5280ctcaacaaag gtgaagagat cacgtctgat accaatatcg aagcggctga agagaatatc 5340tccttggatc tcatccagca atattacctg acctttaact tcgataacga gcccgaaaac 5400atctccatcg agaacctcag ctcagacatc attggtcagt tggagctgat gccaaacatt 5460gaacgcttcc ccaacggcaa gaaatacgaa ctcgacaagt atacgatgtt tcattactta 5520agagcgcagg agtttgaaca cggcaagagc cgcattgctc tcactaactc cgtgaatgaa 5580gccctgctca atccgtcaag ggtgtacaca ttctttagct ccgactatgt caagaaagtg 5640aacaaagcca ccgaagcggc aatgttcctg ggatgggttg aacaactggt ctacgacttc 5700accgacgaga cctctgaggt gagcacaacg gacaagattg ctgacatcac tatcattatc 5760ccgtatattg gacctgcctt gaatattggc aacatgctct acaaagacga tttcgttggt 5820gccctgatct tcagcggtgc cgtgatcctg ttggagttca ttcctgaaat cgccatccct 5880gtgctgggca cgttcgctct ggtctcatac attgcgaata aggtcttgac cgtgcagaca 5940atcgataatg ccctctccaa acgtaacgaa aaatgggacg aggtctacaa atacatcgtg 6000accaactggc tggcaaaggt taacacccaa attgatctga tccgtaagaa aatgaaggag 6060gctttggaga accaggctga agctactaaa gccattatca actaccagta taatcagtat 6120acagaagagg aaaagaataa catcaatttc aacatcgatg acttgtcctc aaagctgaac 6180gagtccatca acaaagctat gatcaacatc aacaaattcc tgaatcagtg ctccgtgtct 6240tacctgatga actctatgat cccatacggt gtgaagcgcc tggaggactt cgatgccagc 6300ctgaaagacg cactgctcaa atacatttac gataatcgcg gcactttgat tggccaagtt 6360gaccgtctga aggacaaggt taacaatacc ttgtcaaccg atatcccctt tcaactgtcc 6420aaatacgttg ataaccagcg cttgctctct actttcaccg aatacattaa caacattatc 6480aatacatcaa ttctcaacct gcgctatgag tccaatcatc tgatcgatct gtctcgttac 6540gccagcaaga tcaacattgg cagcaaagtg aacttcgatc cgattgacaa gaaccaaatc 6600cagttgttca acctcgaaag ctccaaaatc gaagtgatcc tgaagaatgc catcgtctac 6660aactccatgt atgaaaattt ctcaacttca ttctggatta gaatcccgaa atacttcaac 6720tcaatctctc tgaataacga atacacgatc attaactgta tggagaataa ctctggttgg 6780aaggtttcct tgaactatgg agaaattatc tggactctgc aagatacgca agagatcaaa 6840cagcgtgtgg tctttaaata cagccagatg attaacatct ctgactacat caacagatgg 6900atctttgtca ccattacaaa caatcgcctg aataactcca aaatctacat caacggtcgt 6960ctgatcgacc agaaacctat ttcaaacctc ggcaacattc atgcttccaa taacatcatg 7020tttaagttgg atggttgccg cgatacccac cgttacatct ggatcaagta tttcaatctg 7080ttcgacaaag aactcaatga gaaagagatc aaagacttgt atgataatca gtcaaactcc 7140ggcattctga aagacttctg gggcgattac ctccagtacg ataagccata ttacatgctg 7200aatctctatg accctaacaa atatgtggac gtgaacaatg tcggtatccg tggctacatg 7260tacctcaaag gaccacgtgg tagcgttatg acaaccaaca tctacctgaa tagctccttg 7320tatcgcggta cgaagttcat tatcaagaag tacgcttcag gcaacaagga caacatcgtg 7380aggaacaatg atcgcgtgta catcaacgtc gtggtgaaga ataaggaata ccgcttggcg 7440accaacgctt ctcaggctgg agttgagaag atcctgagcg ccttggagat cccagacgtt 7500ggcaacctga gccaagtggt tgtgatgaaa agcaagaatg accagggaat caccaacaaa 7560tgcaaaatga acctgcaaga caacaacggc aatgacatcg gtttcatcgg tttccaccag 7620tttaacaata ttgcgaagct ggtcgccagc aactggtaca acaggcagat tgagaggtca 7680tcccgtacct taggatgctc ttgggaattt atccccgtgg acgatggttg gggcgagaga 7740cccctgggcg caggttggtc ccaccctcag ttcgagaagt aatagttaat agataataat 7800agctcgaggc atgcgagctc cctcaggagg cctacgtcga cgagctcact agtcgcggcc 7860gctttcgaat ctagagcctg cagtctcgag gcatgcggta ccaagcttgt cgagaagtac 7920tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc 7980ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt 8040attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 8100tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 8160tggatctgat cactgcttga gcctaggaga tccgaaccag ataagtgaaa tctagttcca 8220aactattttg tcatttttaa ttttcgtatt agcttacgac gctacaccca gttcccatct 8280attttgtcac tcttccctaa ataatcctta aaaactccat ttccacccct cccagttccc 8340aactattttg tccgcccaca gcggggcatt tttcttcctg ttatgttttt aatcaaacat 8400cctgccaact ccatgtgaca aaccgtcatc ttcggctact ttttctctgt cacagaatga 8460aaatttttct gtcatctctt cgttattaat gtttgtaatt gactgaatat caacgcttat 8520ttgcagcctg aatggcgaat ggThis nucleotide sequence encodes the neurotoxin ΔLC-GFP-BoNT/A^(tev).

Nucleic acid molecules of the present invention also include nucleicacid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% identical to the nucleic acid molecules of SEQ ID NOs:10-12 and15.

Nucleic acid molecules of the present invention may encode the aminoacid sequences of FIG. 1. In particular, the nucleic acid molecules ofthe present invention are modified from the wild type BoNT serotypesequences to have one or more characteristics shown in FIG. 1. Othermodifications may include, without limitation, a mutation which rendersthe encoded propeptide resistant to low-specificity proteolysis, one ormore silent mutations that inactivate putative internal DNA regulatoryelements, and/or one or more unique restriction sites. Mature neurotoxinstability and yield may be optimized by site-directed mutation ofresidues within the intermediate region of the propeptide, therebyreducing the propeptides' susceptibility to non-specific proteolysis andpoisoning of the host organism used for expression of the matureneurotoxin. Also, silent mutations are introduced into DNA regulatoryelements that can affect RNA transcription or expression of thepropeptides in the expression system of choice.

A nucleic acid molecule of the present invention may have a disablingmutation in a region encoding an active metalloprotease site of thepropeptide.

A nucleic acid molecule of the present invention may also have amutation in a region encoding the light chain region, such that thenucleic acid molecule encodes, in the light chain region, a non-nativemotif capable of inactivating light chain metalloprotease activity in aneuron intoxicated by wt Clostridium botulinum neurotoxin.

A nucleic acid molecule of the present invention may have a mutationencoding one or more of the following mutations in the neurotoxin:E₂₂₄>A, Y₃₆₆>A, K₄₃₈>H, K₄₄₀>Q, K₄₄₄>Q, and K₈₇₁>N.

A further aspect of the present invention relates to an expressionsystem having a nucleic acid molecule encoding an isolated Clostridiumbotulinum neurotoxin propeptide of the present invention in aheterologous vector.

Yet another aspect of the present invention relates to a host cellhaving a heterologous nucleic acid molecule encoding an isolatedClostridium botulinum neurotoxin propeptide of the present invention.

Still another aspect of the present invention relates to a method ofexpressing a recombinant physiologically active Clostridium botulinumneurotoxin of the present invention. This method involves providing anucleic acid construct having a nucleic acid molecule encoding anisolated Clostridium botulinum neurotoxin propeptide of the presentinvention. The nucleic acid construct has a heterologous promoteroperably linked to the nucleic acid molecule and a 3′ regulatory regionoperably linked to the nucleic acid molecule. The nucleic acid constructis then introduced into a host cell under conditions effective toexpress the physiologically active Clostridium botulinum neurotoxin.

In a preferred embodiment, the expressed neurotoxin is contacted with ahighly specific protease under conditions effective to effect cleavageat the intermediate region. Preferably, the intermediate region of theClostridium botulinum neurotoxin propeptide is not cleaved by proteasesendogenous to the expression system or the host cell.

Expression of a Botulinum neurotoxin of the present invention can becarried out by introducing a nucleic acid molecule encoding a Botulinumneurotoxin propeptide into an expression system of choice usingconventional recombinant technology. Generally, this involves insertingthe nucleic acid molecule into an expression system to which themolecule is heterologous (i.e., not normally present). The introductionof a particular foreign or native gene into a mammalian host isfacilitated by first introducing the gene sequence into a suitablenucleic acid vector. “Vector” is used herein to mean any geneticelement, such as a plasmid, phage, transposon, cosmid, chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which is capable of transferringgene sequences between cells. Thus, the term includes cloning andexpression vectors, as well as viral vectors. The heterologous nucleicacid molecule is inserted into the expression system or vector in propersense (5′→3′) orientation and correct reading frame. The vector containsthe necessary elements for the transcription and translation of theinserted Botulinum neurotoxin propeptide-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference in its entirety, describes the production of expressionsystems in the form of recombinant plasmids using restriction enzymecleavage and ligation with DNA ligase. These recombinant plasmids arethen introduced by means of transformation and replicated in unicellularcultures including prokaryotic organisms and eukaryotic cells grown intissue culture.

Recombinant genes may also be introduced into viruses, includingvaccinia virus, adenovirus, and retroviruses, including lentivirus.Recombinant viruses can be generated by transfection of plasmids intocells infected with virus.

Suitable vectors include, but are not limited to, the following viralvectors such as lambda vector system gt11, gt WES.tB, Charon 4, andplasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/−or KS +/− (see “Stratagene Cloning Systems” Catalog (1993) fromStratagene, La Jolla, Calif., which is hereby incorporated by referencein its entirety), pQE, pIH821, pGEX, pFastBac series (Invitrogen), pETseries (Studier et. al., “Use of T7 RNA Polymerase to Direct Expressionof Cloned Genes,” Gene Expression Technology Vol. 185 (1990), which ishereby incorporated by reference in its entirety), and any derivativesthereof. Recombinant molecules can be introduced into cells viatransformation, particularly transduction, conjugation, mobilization, orelectroporation. The DNA sequences are cloned into the vector usingstandard cloning procedures in the art, as described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, ColdSprings Harbor, N.Y. (1989), which is hereby incorporated by referencein its entirety.

A variety of host-vector systems may be utilized to express theBotulinum neurotoxin propeptide-encoding sequence in a cell. Primarily,the vector system must be compatible with the host cell used.Host-vector systems include, but are not limited to, the following:bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA;microorganisms such as yeast containing yeast vectors; mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g., baculovirus); and plantcells infected by bacteria. The expression elements of these vectorsvary in their strength and specificities. Depending upon the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements can be used.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation).

Transcription of DNA is dependent upon the presence of a promoter whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eukaryotic promotersdiffer from those of prokaryotic promoters. Furthermore, eukaryoticpromoters and accompanying genetic signals may not be recognized in ormay not function in a prokaryotic system and, further, prokaryoticpromoters are not recognized and do not function in eukaryotic cells.

Similarly, translation of mRNA in prokaryotes depends upon the presenceof the proper prokaryotic signals which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expressionsee Roberts and Lauer, Methods in Enzymology 68:473 (1979), which ishereby incorporated by reference in its entirety.

Promoters vary in their “strength” (i.e., their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promoters in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promoters such as the PH promoter, T7 phage promoter, lacpromoter, trp promoter, recA promoter, ribosomal RNA promoter, the P_(R)and P_(L) promoters of coliphage lambda and others, including but notlimited, to lacUV5, ompF, bla, lpp, and the like, may be used to directhigh levels of transcription of adjacent DNA segments. Additionally, ahybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promoter unless specifically induced. Incertain operons, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in prokaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ tothe initiation codon (ATG) to provide a ribosome binding site. Thus, anySD-ATG combination that can be utilized by host cell ribosomes may beemployed. Such combinations include but are not limited to the SD-ATGcombination from the cro gene or the N gene of coliphage lambda, or fromthe E. coli tryptophan E, D, C, B, or A genes. Additionally, any SD-ATGcombination produced by recombinant DNA or other techniques involvingincorporation of synthetic nucleotides may be used.

Depending on the vector system and host utilized, any number of suitabletranscription and/or translation elements, including constitutive,inducible, and repressible promoters, as well as minimal 5′ promoterelements may be used.

The Botulinum neurotoxin-encoding nucleic acid, a promoter molecule ofchoice, a suitable 3′ regulatory region, and if desired, a reportergene, are incorporated into a vector-expression system of choice toprepare a nucleic acid construct using standard cloning procedures knownin the art, such as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor: Cold Spring HarborLaboratory Press, New York (2001), which is hereby incorporated byreference in its entirety.

The nucleic acid molecule encoding a Botulinum neurotoxin is insertedinto a vector in the sense (i.e., 5→3′) direction, such that the openreading frame is properly oriented for the expression of the encodedBotulinum neurotoxin propeptide under the control of a promoter ofchoice. Single or multiple nucleic acids may be ligated into anappropriate vector in this way, under the control of a suitablepromoter, to prepare a nucleic acid construct.

Once the isolated nucleic acid molecule encoding the Botulinumneurotoxin propeptide has been inserted into an expression vector, it isready to be incorporated into a host cell. Recombinant molecules can beintroduced into cells via transformation, particularly transduction,conjugation, lipofection, protoplast fusion, mobilization, particlebombardment, or electroporation. The DNA sequences are incorporated intothe host cell using standard cloning procedures known in the art, asdescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y.(1989), which is hereby incorporated by reference in its entirety.Suitable hosts include, but are not limited to, bacteria, virus, yeast,fungi, mammalian cells, insect cells, plant cells, and the like.Preferable host cells of the present invention include, but are notlimited to, Escherichia coli, insect cells, and Pichia pastoris cells.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent in the plasmid with which the host cell was transformed.Suitable genes are those which confer resistance to gentamycin, G418,hygromycin, puromycin, streptomycin, spectinomycin, tetracycline,chloramphenicol, and the like. Similarly, “reporter genes” which encodeenzymes providing for production of an identifiable compound, or othermarkers which indicate relevant information regarding the outcome ofgene delivery, are suitable. For example, various luminescent orphosphorescent reporter genes are also appropriate, such that thepresence of the heterologous gene may be ascertained visually.

In one embodiment of the present invention, the expressed neurotoxinpropeptide is contacted with a highly specific protease (e.g.,enterokinase or TEV sequence) under conditions effective to enablecleavage at the intermediate region of the propeptide of the presentinvention. By this means, the intermediate region is not cleaved byproteases endogenous to the host cell. The expressed neurotoxinpropeptide has one or more disulfide bridges.

Another aspect of the present invention relates to an isolated,physiologically active Clostridium botulinum neurotoxin produced bycleaving an isolated Clostridium botulinum neurotoxin propeptide of thepresent invention. The propeptide is cleaved at the highly specificprotease cleavage site. The light and heavy chain regions are linked bya disulfide bond.

The Clostridium botulinum neurotoxin of the present invention can beisolated at a yield or concentration of at least about 0.1 mg/L, atleast about 0.5 mg/L, at least about 1 mg/L, at least about 5 mg/L, atleast about 10 mg/L, about 10-20 mg/L, about 20-30 mg/L, or at leastabout 30 mg/L. One of the particular advantages of the propeptides ofthe present invention and the method of their expression describedherein is that BoNT neurotoxins can be purified to a homogeneity using atwo-stage, non-denaturing, and highly selective affinity purification,as described in greater detail infra.

As discussed supra, Botulinum neurotoxins are synthesized as singlechain propeptides which are later activated by a specific proteolysiscleavage event, generating a dimer joined by a disulfide bond. Thesestructural features can be illustrated using BoNT/A as an example, andare generally applicable to all Clostridium botulinum serotypes. Themature BoNT/A is composed of three functional domains of Mr ˜50,000,where the catalytic function responsible for toxicity is confined to thelight chain (residues 1-437), the translocation activity is associatedwith the N-terminal half of the heavy chain (residues 448-872), and cellbinding is associated with its C-terminal half (residues 873-1,295)(Johnson, “Clostridial Toxins as Therapeutic Agents: Benefits ofNature's Most Toxic Proteins,” Annu. Rev. Microbiol. 53:551-575 (1999);Montecucco et al., “Structure and Function of Tetanus and BotulinumNeurotoxins,” Q. Rev. Biophys. 28:423-472 (1995), which are herebyincorporated by reference in their entirety).

Optimized expression and recovery of recombinant neurotoxins for BoNTserotypes in a native and physiologically active state is achieved bythe introduction of one or more alterations to the nucleotide sequencesencoding the BoNT propeptides, as discussed supra. These mutations aredesigned to maximize yield of recombinant Botulinum neurotoxin, whileretaining the native toxins' structure and biological activity.

Isolated, full-length Clostridium botulinum neurotoxins of the presentinvention are physiologically active. This physiological activityincludes, but is not limited to, toxin immunogenicity, trans- andintra-cellular trafficking, and cell recognition.

The mechanism of cellular binding and internalization of Clostridialtoxins is still poorly understood. No specific receptor has beenunambiguously identified, and the binding constants have not beencharacterized. The C-terminal portion of the heavy chain of allBotulinum neurotoxins binds to gangliosides (sialic acid-containingglycolipids), with a preference for gangliosides of the G_(1b) series(Montecucco et al., “Structure and Function of Tetanus and BotulinumNeurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Montecucco, “How DoTetanus and Botulinum Toxins Bind to Neuronal Membranes?” TIBS11:314-317 (1986); and Van Heyningen et al., “The Fixation of TetanusToxin by Ganglioside,” J. Gen. Microbiol. 24:107-119 (1961), which arehereby incorporated by reference in their entirety). The sequenceresponsible for ganglioside binding has been identified for thestructurally similar TeNT molecule, and is located within the 34C-terminal amino acid residues of its heavy chain. BoNT/A, /B, /C, /E,and /F share a high degree of homology with TeNT in this region (Shapiroet al., “Identification of a Ganglioside Recognition Domain of TetanusToxin Using a Novel Ganglioside Photoaffinity Ligand,” J. Biol. Chem.272:30380-30386 (1997), which is hereby incorporated by reference in itsentirety). Multiple types of evidence suggest the existence of at leastone additional component involved in the binding of Botulinumneurotoxins to neuronal membranes (Montecucco et al., “Structure andFunction of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys.28:423-472 (1995); Montecucco, “How Do Tetanus and Botulinum Toxins Bindto Neuronal Membranes?” TIBS 11:314-317 (1986), which are herebyincorporated by reference in their entirety). In two reports (Nishiki etal., “The High-Affinity Binding of Clostridium Botulinum Type BNeurotoxin to Synaptotagmin II Associated with GangliosidesG_(T1b)/G_(D1a) ,” FEBS Lett. 378:253-257 (1996); Dong et al.,“Synaptotagmins I and II Mediate Entry of Botulinum Neurotoxin B intoCells,” J. Cell Biol. 162:1293-1303 (2003), which are herebyincorporated by reference in their entirety), synaptotagmins wereidentified as possible candidates for the auxiliary BoNT/B receptor, andsynaptotagmins I and II were implicated as neuronal receptors for BoNT/G(Rummel et al., “Synaptotagmins I and II Act as Nerve Cell Receptors forBotulinum Neurotoxin G,” J. Biol. Chem. 279:30865-30870 (2004), which ishereby incorporated by reference in its entirety). However, despite thestructural similarity in the putative receptor-binding domain ofBotulinum neurotoxins, other toxin subtypes show no affinity forsynaptotagmins or synaptotagmin-related molecules. Lipid rafts (Herreroset al., “Lipid Rafts Act as Specialized Domains for Tetanus ToxinBinding and Internalization Into Neurons,” Mol. Biol. Cell 12:2947-2960(2001), which is hereby incorporated by reference in its entirety) havebeen implicated as a specialized domain involved in TeNT binding andinternalization into neurons, but these domains are widely distributedon multiple cell types, and therefore cannot simply explain the highspecificity of the toxins for neurons.

Botulinum neurotoxins are internalized through the presynaptic membraneby an energy-dependent mechanism (Montecucco et al., “Structure andFunction of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys.28:423-472 (1995); Matteoli et al., “Synaptic Vesicle EndocytosisMediates the Entry of Tetanus Neurotoxin into Hippocampal Neurons,”Proc. Natl. Acad. Sci. USA 93:13310-13315 (1996); and Mukherjee et al.,“Endocytosis,” Physiol. Rev. 77:759-803 (1997), which are herebyincorporated by reference in their entirety), and rapidly appear invesicles where they are at least partially protected from degradation(Dolly et al., “Acceptors for Botulinum Neurotoxin Reside on Motor NerveTerminals and Mediate Its Internalization,” Nature 307:457-460 (1984);Critchley et al., “Fate of Tetanus Toxin Bound to the Surface of PrimaryNeurons in Culture: Evidence for Rapid Internalization,” J. Cell Biol.100:1499-1507 (1985), which are hereby incorporated by reference intheir entirety). The BoNT complex of light and heavy chains interactswith the endocytic vesicle membrane in a chaperone-like way, preventingaggregation and facilitating translocation of the light chain in afashion similar to the protein conducting/translocating channels ofsmooth ER, mitochondria, and chloroplasts (Koriazova et al.,“Translocation of Botulinum Neurotoxin Light Chain Protease Through theHeavy Chain Channel,” Nat. Struct. Biol. 10:13-18 (2003), which ishereby incorporated by reference in its entirety). Acidification of theendosome is believed to induce pore formation, which allowstranslocation of the light chain to the cytosol upon reduction of theinterchain disulfide bond (Hoch et al., “Channels Formed by Botulinum,Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance toTranslocation of Proteins Across Membranes,” Proc. Natl. Acad. Sci. USA82:1692-1696 (1985), which is hereby incorporated by reference in itsentirety). Within the cytosol, the light chain displays azinc-endopeptidase activity specific for protein components of thesynaptic vesicle exocytosis apparatus. TeNT and BoNT/B, /D, /F, and /Grecognize VAMP/synaptobrevin. This integral protein of the synapticvesicle membrane is cleaved at a single peptide bond, which differs foreach neurotoxin. BoNT/A, /C, and /E recognize and cleave SNAP-25, aprotein of the presynaptic membrane, at two different sites within thecarboxyl terminus. BoNT/C also cleaves syntaxin, another protein of thenerve plasmalemma (Montecucco et al., “Structure and Function of Tetanusand Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Suttonet al., “Crystal Structure of a SNARE Complex Involved in SynapticExocytosis at 2.4 {acute over (Å)} Resolution,” Nature 395:347-353(1998), which are hereby incorporated by reference in their entirety).The cleavage of any component of the synaptic release machinery resultsin inhibition of acetylcholine release, ultimately leading toneuromuscular paralysis.

The toxicity of Botulinum neurotoxins is a result of a multi-stepmechanism. From the circulation, BoNT targets the pre-synaptic membraneof neuromuscular junctions, where it is internalized to directly exertits toxic effect on the peripheral nervous system (Dolly et al.,“Acceptors for Botulinum Neurotoxin Reside on Motor Nerve Terminals andMediate Its Internalization,” Nature 307 :457-460 (1984), which ishereby incorporated by reference in its entirety). Toxicity at theneuromuscular junction involves neuron binding; internalization intoendocytic vesicles, similar to those involved in synaptic vesiclerecycling; activation within an acidic compartment to theproteolytically active toxin which then penetrates into the neuronalcytoplasm; and target recognition and catalytic cleavage of substratesin the neuronal machinery for synaptic vesicle exocytosis.

Clostridium botulinum neurotoxins of the present invention arephysiologically active, and may be either toxic or atoxic. Atoxicneurotoxins have a toxicity that is reduced from the wt BoNT by at leastabout 1000-fold. In certain exemplary embodiments, the LD₅₀ of an atoxicClostridium botulinum neurotoxin of the present invention is between1,000 and 150,000; between 50,000 and 150,000; between 75,000 and150,000; between 100,000 and 150,000; between 1,000 and 100,000; between50,000 and 100,000; between 75,000 and 100,000; 1,000; 25,000; 50,000;75,000; 100,000; or 150,000-fold higher than the LD₅₀ of wt Clostridiumbotulinum.

The endopeptidase activity responsible for Botulinum neurotoxin toxicityis believed to be associated with the presence of a HExxHxxH (SEQ IDNO:1) motif in the light chain, characteristic of metalloproteases.Mutagenesis of BoNT/A light chain, followed by microinjection of thecorresponding mRNA into presynaptic cholinergic neurons of Aplysiacalifornica, allowed the minimal essential domain responsible fortoxicity to be identified (Kurazono et al., “Minimal Essential DomainsSpecifying Toxicity of the Light Chains of Tetanus Toxin and BotulinumNeurotoxin Type A,” J. Biol. Chem. 267:14721-14729 (1992), which ishereby incorporated by reference in its entirety). Site-directedmutagenesis of BoNT/A light chain pinpointed the amino acid residuesinvolved in Zn²⁺ coordination, and formation of the activemetalloendoprotease core which cleaves SNAP-25 (Rigoni et al.,“Site-Directed Mutagenesis Identifies Active-Site Residues of the LightChain of Botulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun.288:1231-1237 (2001), which is hereby incorporated by reference in itsentirety). The three-dimensional structures of Botulinum neurotoxins andtheir derivatives confirmed the mutagenesis results, and detailed thespatial organization of the protein domains. For the BoNT/A holotoxin,crystal structure was obtained to a resolution of 3.3 {acute over (Å)}(Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type A andImplications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998), whichis hereby incorporated by reference in its entirety). The BoNT/Bholotoxin crystal structure was determined at 1.8 and 2.6 {acute over(Å)} resolution (Swaminathan et al., “Structural Analysis of theCatalytic and Binding Sites of Clostridium botulinum Neurotoxin B,” Nat.Struct. Biol. 7:693-699 (2000), which is hereby incorporated byreference in its entirety). Recently, a crystal structure for BoNT/Ecatalytic domain was determined to 2.1 {acute over (Å)} resolution(Agarwal et al., “Structural Analysis of Botulinum Neurotoxin Type ECatalytic Domain and Its Mutant Glu₂₁₂>Gln Reveals the Pivotal Role ofthe Glu₂₁₂ Carboxylate in the Catalytic Pathway,” Biochemistry43:6637-6644 (2004), which is hereby incorporated by reference in itsentirety). The later study provided multiple interesting structuraldetails, and helps explain the complete loss of metalloendoproteolyticactivity in the BoNT/E LC E₂₁₂>Q mutant. The availability of thisdetailed information on the relationship between the amino acid sequenceand biological activities of Clostridial toxins enables the design ofmodified toxin genes with properties specifically altered fortherapeutic goals.

Thus, the physiologically active and atoxic Botulinum neurotoxin of thepresent invention may have a disabling mutation in an activemetalloprotease site. The physiologically active Botulinum neurotoxinsof the present invention may also have a non-native motif (e.g., a SNAREmotif) in the light chain region that is capable of inactivating lightchain metalloprotease activity in a toxic Botulinum neurotoxin, asdescribed in U.S. Patent Application Publication No. 2006/0204524 toIchtchenko et al., which is hereby incorporated by reference in itsentirety.

Exemplary Clostridium botulinum neurotoxins of the present inventionhave structures as shown in FIG. 1. Specifically, the neurotoxin mayhave a structure selected from BoNT/Aad^(ek), BoNT/Aad^(tev),ΔLC-Peptide-BoNT/A^(tev), and ΔLC-GFP-BoNT/A^(tev), or other suchsimilar derivatives. Alternatively, neurotoxins of the present inventionhave an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NOs:3-6 set forth below.

The full-length BoNT/Aad^(ek) neurotoxin has an amino acid sequence ofSEQ ID NO:3, as follows:

MKFLVNVALV FMVVYISYIY AAAHHHHHHH HHHTRENLYF QGAGDSLSWL LRLLNARGGA 60SGPFVNKQFN YKDPVNGVDI AYIKIPNAGQ MQPVKAFKIH NKIWVIPERD TFTNPEEGDL 120NPPPEAKQVP VSYYDSTYLS TDNEKDNYLK GVTKLFERIY STDLGRMLLT SIVRGIPFWG 180GSTIDTELKV IDTNCINVIQ PDGSYRSEEL NLVIIGPSAD IIQFECKSFG HEVLNLTRNG 240YGSTQYIRFS PDFTFGFEES LEVDTNPLLG AGKFATDPAV TLAHALIHAG HRLYGIAINP 300NRVFKVNTNA YYEMSGLEVS FEELRTFGGH DAKFIDSLQE NEFRLYYYNK FKDIASTLNK 360AKSIVGTTAS LQYMKNVFKE KYLLSEDTSG KFSVDKLKFD KLYKMLTEIY TEDNFVKFFK 420VLNRKTALNF DKAVFKINIV PKVNYTIYDG FNLRNTNLAA NFNGQNTEIN NMNFTKLKNF 480TGLFEFYKLL CVRGIITSHT QSLDQGYNDD DDKALNDLCI KVNNWDLFFS PSEDNFTNDL 540NKGEEITSDT NIEAAEENIS LDLIQQYYLT FNFDNEPENI SIENLSSDII GQLELMPNIE 600RFPNGKKYEL DKYTMFHYLR AQEFEHGKSR IALTNSVNEA LLNPSRVYTF FSSDYVKKVN 660KATEAAMFLG WVEQLVYDFT DETSEVSTTD KIADITIIIP YIGPALNIGN MLYKDDFVGA 720LIFSGAVILL EFIPEIAIPV LGTFALVSYI ANKVLTVQTI DNALSKRNEK WDEVYKYIVT 780NWLAKVNTQI DLIRKKMKEA LENQAEATKA IINYQYNQYT EEEKNNINFN IDDLSSKLNE 840SINKAMININ KFLNQCSVSY LMNSMIPYGV KRLEDFDASL KDALLKYIYD NRGTLIGQVD 900RLKDKVNNTL STDIPFQLSK YVDNQRLLST FTEYINNIIN TSILNLRYES NHLIDLSRYA 960SKINIGSKVN FDPIDKNQIQ LFNLESSKIE VILKNAIVYN SMYENFSTSF WIRIPKYFNS 1020ISLNNEYTII NCMENNSGWK VSLNYGEIIW TLQDTQEIKQ RVVFKYSQMI NISDYINRWI 1080FVTITNNRLN NSKIYINGRL IDQKPISNLG NIHASNNIMF KLDGCRDTHR YIWIKYFNLF 1140DKELNEKEIK DLYDNQSNSG ILKDFWGDYL QYDKPYYMLN LYDPNKYVDV NNVGIRGYMY 1200LKGPRGSVMT TNIYLNSSLY RGTKFIIKKY ASGNKDNIVR NNDRVYINVV VKNKEYRLAT 1260NASQAGVEKI LSALEIPDVG NLSQVVVMKS KNDQGITNKC KMNLQDNNGN DIGFIGFHQF 1320NNIAKLVASN WYNRQIERSS RTLGCSWEFI PVDDGWGERP LGAGWSHPQF EK

The full-length BoNT/Aad^(tev) neurotoxin has an amino acid sequence ofSEQ ID NO:4, as follows:

MKFLVNVALV FMVVYISYIY AAAHHHHHHH HHHTRENLYF QGAGDSLSWL LRLLNARGGA 60SGPFVNKQFN YKDPVNGVDI AYIKIPNAGQ MQPVKAFKIH NKIWVIPERD TFTNPEEGDL 120NPPPEAKQVP VSYYDSTYLS TDNEKDNYLK GVTKLFERIY STDLGRMLLT SIVRGIPFWG 180GSTIDTELKV IDTNCINVIQ PDGSYRSEEL NLVIIGPSAD IIQFECKSFG HEVLNLTRNG 240YGSTQYIRFS PDFTFGFEES LEVDTNPLLG AGKFATDPAV TLAHALIHAG HRLYGIAINP 300NRVFKVNTNA YYEMSGLEVS FEELRTFGGH DAKFIDSLQE NEFRLYYYNK FKDIASTLNK 360AKSIVGTTAS LQYMKNVFKE KYLLSEDTSG KFSVDKLKFD KLYKMLTEIY TEDNFVKFFK 420VLNRKTALNF DKAVFKINIV PKVNYTIYDG FNLRNTNLAA NFNGQNTEIN NMNFTKLKNF 480TGLFEFYKLL CVRGIITSHT QSLDQGGENL YFQGALNDLC IKVNNWDLFF SPSEDNFTND 540LNKGEEITSD TNIEAAEENI SLDLIQQYYL TFNFDNEPEN ISIENLSSDI IGQLELMPNI 600ERFPNGKKYE LDKYTMFHYL RAQEFEHGKS RIALTNSVNE ALLNPSRVYT FFSSDYVKKV 660NKATEAAMFL GWVEQLVYDF TDETSEVSTT DKIADITIII PYIGPALNIG NMLYKDDFVG 720ALIFSGAVIL LEFIPEIAIP VLGTFALVSY IANKVLTVQT IDNALSKRNE KWDEVYKYIV 780TNWLAKVNTQ IDLIRKKMKE ALENQAEATK AIINYQYNQY TEEEKNNINF NIDDLSSKLN 840ESINKAMINI NKFLNQCSVS YLMNSMIPYG VKRLEDFDAS LKDALLKYIY DNRGTLIGQV 900DRLKDKVNNT LSTDIPFQLS KYVDNQRLLS TFTEYINNII NTSILNLRYE SNHLIDLSRY 960ASKINIGSKV NFDPIDKNQI QLFNLESSKI EVILKNAIVY NSMYENFSTS FWIRIPKYFN 1020SISLNNEYTI INCMENNSGW KVSLNYGEII WTLQDTQEIK QRVVFKYSQM INISDYINRW 1080IFVTITNNRL NNSKIYINGR LIDQKPISNL GNIHASNNIM FKLDGCRDTH RYIWIKYFNL 1140FDKELNEKEI KDLYDNQSNS GILKDFWGDY LQYDKPYYML NLYDPNKYVD VNNVGIRGYM 1200YLKGPRGSVM TTNIYLNSSL YRGTKFIIKK YASGNKDNIV RNNDRVYINV VVKNKEYRLA 1260TNASQAGVEK ILSALEIPDV GNLSQVVVMK SKNDQGITNK CKMNLQDNNG NDIGFIGFHQ 1320FNNIAKLVAS NWYNRQIERS SRTLGCSWEF IPVDDGWGER PLGAGWSHPQ FEK

The full-length ΔLC-Peptide-BoNT/A^(tev) neurotoxin has an amino acidsequence of SEQ ID NO:5, as follows:

MKFLVNVALV FMVVYISYIY AAAHHHHHHH HHHTRENLYF QGAGDSLSWL LRLLNARGGA 60SGGTNGNGNG GNLRNTNLAA NFNGQNTEIN NMNFTKLKNF TGLFEFYKLL CVRGIITSHT 120QSLDQGGENL YFQGALNDLC IKVNNWDLFF SPSEDNFTND LNKGEEITSD TNIEAAEENI 180SLDLIQQYYL TFNFDNEPEN ISIENLSSDI IGQLELMPNI ERFPNGKKYE LDKYTMFHYL 240RAQEFEHGKS RIALTNSVNE ALLNPSRVYT FFSSDYVKKV NKATEAAMFL GWVEQLVYDF 300TDETSEVSTT DKIADITIII PYIGPALNIG NMLYKDDFVG ALIFSGAVIL LEFIPEIAIP 360VLGTFALVSY IANKVLTVQT IDNALSKRNE KWDEVYKYIV TNWLAKVNTQ IDLIRKKMKE 420ALENQAEATK AIINYQYNQY TEEEKNNINF NIDDLSSKLN ESINKAMINI NKFLNQCSVS 480YLMNSMIPYG VKRLEDFDAS LKDALLKYIY DNRGTLIGQV DRLKDKVNNT LSTDIPFQLS 540KYVDNQRLLS TFTEYINNII NTSILNLRYE SNHLIDLSRY ASKINIGSKV NFDPIDKNQI 600QLFNLESSKI EVILKNAIVY NSMYENFSTS FWIRIPKYFN SISLNNEYTI INCMENNSGW 660KVSLNYGEII WTLQDTQEIK QRVVFKYSQM INISDYINRW IFVTITNNRL NNSKIYINGR 720LIDQKPISNL GNIHASNNIM FKLDGCRDTH RYIWIKYFNL FDKELNEKEI KDLYDNQSNS 780GILKDFWGDY LQYDKPYYML NLYDPNKYVD VNNVGIRGYM YLKGPRGSVM TTNIYLNSSL 840YRGTKFIIKK YASGNKDNIV RNNDRVYINV VVKNKEYRLA TNASQAGVEK ILSALEIPDV 900GNLSQVVVMK SKNDQGITNK CKMNLQDNNG NDIGFIGFHQ FNNIAKLVAS NWYNRQIERS 960SRTLGCSWEF IPVDDGWGER PLGAGWSHPQ FEK

The full-length ΔLC-GFP-BoNT/A^(tev) neurotoxin has an amino acidsequence of SEQ ID NO:6, as follows:

MKFLVNVALV FMVVYISYIY AAAHHHHHHH HHHTRENLYF QGAGDSLSWL LRLLNARGGA 60SVSKGAELFT GIVPILIELN GDVNGHKFSV SGEGEGDATY GKLTLKFICT TGKLPVPWPT 120LVTTLSYGVQ CFSRYPDHMK QHDFFKSAMP EGYIQERTIF FEDDGNYKSR AEVKFEGDTL 180VNRIELTGTD FKEDGNILGN KMEYNYNAHN VYIMTDKAKN GIKVNFKIRH NIEDGSVQLA 240DHYQQNTPIG DGPVLLPDNH YLSTQSALSK DPNEKRDHMI YFGFVTAAAI THGMDELYKG 300TNGNGNGGNL RNTNLAANFN GQNTEINNMN FTKLKNFTGL FEFYKLLCVR GIITSHTQSL 360DQGGENLYFQ GALNDLCIKV NNWDLFFSPS EDNFTNDLNK GEEITSDTNI EAAEENISLD 420LIQQYYLTFN FDNEPENISI ENLSSDIIGQ LELMPNIERF PNGKKYELDK YTMFHYLRAQ 480EFEHGKSRIA LTNSVNEALL NPSRVYTFFS SDYVKKVNKA TEAAMFLGWV EQLVYDFTDE 540TSEVSTTDKI ADITIIIPYI GPALNIGNML YKDDFVGALI FSGAVILLEF IPEIAIPVLG 600TFALVSYIAN KVLTVQTIDN ALSKRNEKWD EVYKYIVTNW LAKVNTQIDL IRKKMKEALE 660NQAEATKAII NYQYNQYTEE EKNNINFNID DLSSKLNESI NKAMININKF LNQCSVSYLM 720NSMIPYGVKR LEDFDASLKD ALLKYIYDNR GTLIGQVDRL KDKVNNTLST DIPFQLSKYV 780DNQRLLSTFT EYINNIINTS ILNLRYESNH LIDLSRYASK INIGSKVNFD PIDKNQIQLF 840NLESSKIEVI LKNAIVYNSM YENFSTSFWI RIPKYFNSIS LNNEYTIINC MENNSGWKVS 900LNYGEIIWTL QDTQEIKQRV VFKYSQMINI SDYINRWIFV TITNNRLNNS KIYINGRLID 960QKPISNLGNI HASNNIMFKL DGCRDTHRYI WIKYFNLFDK ELNEKEIKDL YDNQSNSGIL 1020KDFWGDYLQY DKPYYMLNLY DPNKYVDVNN VGIRGYMYLK GPRGSVMTTN IYLNSSLYRG 1080TKFIIKKYAS GNKDNIVRNN DRVYINVVVK NKEYRLATNA SQAGVEKILS ALEIPDVGNL 1140SQVVVMKSKN DQGITNKCKM NLQDNNGNDI GFIGFHQFNN IAKLVASNWY NRQIERSSRT 1200LGCSWEFIPV DDGWGERPLG AGWSHPQFEK

Still another aspect of the present invention relates to a treatmentmethod. This method involves providing an isolated Clostridium botulinumneurotoxin according to the present invention, where the light chainregion and the heavy chain region are linked by a disulfide bond and thecargo comprises a therapeutic agent. The isolated Clostridium botulinumneurotoxin is administered to an individual in need of treatment underconditions effective to provide treatment to the individual.

Administration can be carried out orally, parenterally, for example,subcutaneously, intravenously, intramuscularly, intrarticularly,intraperitoneally, by intranasal instillation, or by application tomucous membranes, such as, that of the nose, throat, and bronchialtubes. The neurotoxin may be administered alone or with suitablepharmaceutical carriers, and can be in solid or liquid form such as,tablets, capsules, powders, solutions, suspensions, or emulsions.

The neurotoxin of the present invention may be orally administered, forexample, with an inert diluent, or with an assimilable edible carrier,or may be enclosed in hard or soft shell capsules, or may be compressedinto tablets, or may be incorporated directly with the food of the diet.For oral therapeutic administration, the neurotoxin (along with anycargo) may be incorporated with excipients and used in the form oftablets, capsules, elixirs, suspensions, syrups, and the like. Suchcompositions and preparations should contain at least 0.1% of activecompound. The percentage of the compound in these compositions may, ofcourse, be varied and may conveniently be between about 2% to about 60%of the weight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions according to the present inventionare prepared so that an oral dosage unit contains between about 1 and250 mg of active compound.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor.

The neurotoxin may also be administered parenterally. Solutions orsuspensions can be prepared in water suitably mixed with a surfactant,such as hydroxypropylcellulose. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof in oils.Illustrative oils are those of petroleum, animal, vegetable, orsynthetic origin, for example, peanut oil, soybean oil, or mineral oil.In general, water, saline, aqueous dextrose and related sugar solution,and glycols such as, propylene glycol, hyaluronan and its derivatives,or polyethylene glycol, are preferred liquid carriers, particularly forinjectable solutions. Under ordinary conditions of storage and use,these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

The neurotoxin of the present invention may also be administereddirectly to the airways in the form of an aerosol. For use as aerosols,the neurotoxin of the present invention in solution or suspension may bepackaged in a pressurized aerosol container together with suitablepropellants, for example, hydrocarbon propellants like propane, butane,or isobutane with conventional adjuvants. The neurotoxin of the presentinvention also may be administered in a non-pressurized form such as ina nebulizer or atomizer.

In one embodiment, the neurotoxin of the present invention has atherapeutic agent as cargo, and the method involves treatment by thetherapeutic agent. According to this embodiment, the Botulinumneurotoxin serves as a delivery vehicle for the therapeutic agent.

In another embodiment, the neurotoxin of the present invention has cargoother than a therapeutic agent and is administered to treat disordersamenable to treatment by Botulinum neurotoxins (either toxic or atoxic)including, for example, neuronal pathologies, such as tourettes syndrome(Porta et al., “Treatment of Phonic Tics in Patients with Tourette'sSyndrome Using Botulinum Toxin Type A,” Neurol. Sci. 24:420-423 (2004),which is hereby incorporated by reference in its entirety) and focalmuscle spasticity or dystonias (MacKinnon et al., “CorticospinalExcitability Accompanying Ballistic Wrist Movements in PrimaryDystonia,” Mov. Disord. 19:273-284 (2004), which is hereby incorporatedby reference in its entirety) including, but not limited to, treatmentfor cervical dystonia (Haussermann et al., “Long-Term Follow-Up ofCervical Dystonia Patients Treated with Botulinum Toxin A,” Mov. Disord.19:303-308 (2004), which is hereby incorporated by reference in itsentirety), primary blepharospasm (Defazio et al., “PrimaryBlepharospasm: Diagnosis and Management,” Drugs 64:237-244 (2004), whichis hereby incorporated by reference in its entirety), hemifacial spasm,post-stroke (Bakheit, “Optimising the Methods of Evaluation of theEffectiveness of Botulinum Toxin Treatment of Post-Stroke MuscleSpasticity,” J. Neurol. Neurosurg. Psychiatry 75:665-666 (2004), whichis hereby incorporated by reference in its entirety), spasmodicdysphonia (Bender et al., “Speech Intelligibility in Severe AdductorSpasmodic Dysphonia,” J. Speech Lang. Hear Res. 47:21-32 (2004), whichis hereby incorporated by reference in its entirety), facial nervedisorders (Finn, “Botulinum Toxin Type A: Fine-Tuning Treatment ofFacial Nerve Injury,” J. Drugs Dermatol. 3:133-137 (2004), which ishereby incorporated by reference in its entirety), and Rasmussensyndrome (Lozsadi et al., “Botulinum Toxin A Improves Involuntary LimbMovements in Rasmussen Syndrome,” Neurology 62:1233-1234 (2004), whichis hereby incorporated by reference in its entirety). Other neurologictreatments include treatment for amputation pain (Kern et al., “Effectsof Botulinum Toxin Type B on Stump Pain and Involuntary Movements of theStump,” Am. J. Phys. Med. Rehabil. 83:396-399 (2004), which is herebyincorporated by reference in its entirety), voice tremor (Adler et al.,“Botulinum Toxin Type A for Treating Voice Tremor,” Arch. Neurol.61:1416-1420 (2004), which is hereby incorporated by reference in itsentirety), crocodile tear syndrome (Kyrmizakis et al., “The Use ofBotulinum Toxin Type A in the Treatment of Frey and Crocodile TearsSyndrome,” J. Oral Maxillofac. Surg. 62:840-844 (2004), which is herebyincorporated by reference in its entirety), marginal mandibular nerveparalysis, and pain control (Cui et al., “Subcutaneous Administration ofBotulinum Toxin A Reduces Formalin-Induced Pain,” Pain 107:125-133(2004), which is hereby incorporated by reference in its entirety)including, but not limited to, pain after mastectomy (Layeeque et al.,“Botulinum Toxin Infiltration for Pain Control After Mastectomy andExpander Reconstruction,” Ann. Surg. 240:608-613 (2004), which is herebyincorporated by reference in its entirety) and chest pain of esophagealorigin (Schumulson et al., “Current and Future Treatment of Chest Painof Presumed Esophageal Origin,” Gastroenterol. Clin. North Am. 33:93-105(2004), which is hereby incorporated by reference in its entirety).Another neurologic treatment amenable to the methods of the presentinvention is headache (Blumenfeld et al., “Botulinum Neurotoxin for theTreatment of Migraine and Other Primary Headache Disorders,” Dermatol.Clin. 22:167-175 (2004), which is hereby incorporated by reference inits entirety).

The methods of the present invention are also suitable for treatment ofcerebral palsy (Balkrishnan et al., “Longitudinal Examination of HealthOutcomes Associated with Botulinum Toxin Use in Children with CerebralPalsy,” J. Surg. Orthop. Adv. 13:76-80 (2004); Berweck et al., “Use ofBotulinum Toxin in Pediatric Spasticity (Cerebral Palsy),” Mov. Disord.19:S162-S167 (2004); Pidcock, “The Emerging Role of TherapeuticBotulinum Toxin in the Treatment of Cerebral Palsy,” J. Pediatr.145:S33-S35 (2004), which are hereby incorporated by reference in theirentirety), hip adductor muscle dysfunction in multiple sclerosis (Wisselet al., “Botulinum Toxin Treatment of Hip Adductor Spasticity inMultiple Sclerosis,” Wien Klin Wochesnchr 4:20-24 (2001), which ishereby incorporated by reference in its entirety), neurogenic pain andinflammation, including arthritis, iatrogenic parotid sialocele(Capaccio et al., “Diagnosis and Therapeutic Management of IatrogenicParotid Sialocele,” Ann. Otol. Rhinol. Laryngol. 113:562-564 (2004),which is hereby incorporated by reference in its entirety), and chronicTMJ displacement (Aquilina et al., “Reduction of a Chronic BilateralTemporomandibular Joint Dislocation with Intermaxillary Fixation andBotulinum Toxin A,” Br. J. Oral Maxillofac. Surg. 42:272-273 (2004),which is hereby incorporated by reference in its entirety). Otherconditions that can be treated by local controlled delivery ofpharmaceutically active toxin include intra-articular administration forthe treatment of arthritic conditions (Mahowald et al., “Long TermEffects of Intra-Articular BoNT A for Refractory Joint Pain,” AnnualMeeting of the American College of Rheumatology (2004), which is herebyincorporated by reference in its entirety), and local administration forthe treatment of joint contracture (Russman et al., “Cerebral Palsy: ARational Approach to a Treatment Protocol, and the Role of BotulinumToxin in Treatment,” Muscle Nerve Suppl. 6:S181-S193 (1997); Pucinelliet al., “Botulinic Toxin for the Rehabilitation of OsteoarthritisFixed-Flexion Knee Deformity,” Annual Meeting of the OsteoartritisResearch Society International (2004), which are hereby incorporated byreference in their entirety). The methods of the present invention arealso suitable for the treatment of pain associated with variousconditions characterized by the sensitization of nociceptors and theirassociated clinical syndromes, as described in Bach-Rojecky et al.,“Antinociceptive Effect of Botulinum Toxin Type A In Rat Model ofCarrageenan and Capsaicin Induced Pain,” Croat. Med. J. 46:201-208(2005); Aoki, “Evidence for Antinociceptive Activity of Botulinum ToxinType A in Pain Management,” Headache 43 Suppl 1:S9-15 (2003); Kramer etal., “Botulinum Toxin A Reduces Neurogenic Flare But Has Almost NoEffect on Pain and Hyperalgesia in Human Skin,” J. Neurol. 250:188-193(2003); Blersch et al., “Botulinum Toxin A and the Cutaneous Nociceptionin Humans: A Prospective, Double-Blind, Placebo-Controlled, RandomizedStudy,” J. Neurol. Sci. 205:59-63 (2002), which are hereby incorporatedby reference in their entirety.

In one embodiment, accumation of atoxic Botulinum neurotoxins of thepresent invention at particular sites (e.g., in neuronal cells inperiarticular tissues of joints) may be effective in treating disordersdescribed herein (e.g., arthritis and chronic musculoskeletal painsyndromes, and other disorders associated with the central painpathway). Accumulation may include accumulation of the light chain inneuronal cytosol.

The methods and products of the present invention may be customized tooptimize therapeutic properties (see e.g., Chaddock et al., “RetargetedClostridial Endopeptidases: Inhibition of Nociceptive NeurotransmitterRelease In vitro, and Antinociceptive Activity in In vivo Models ofPain,” Mov. Disord. 8:S42-S47 (2004); Finn, “Botulinum Toxin Type A:Fine-Tuning Treatment of Facial Nerve Injury,” J. Drugs Dermatol.3:133-137 (2004); Eleopra et al., “Different Types of Botulinum Toxin InHumans,” Mov. Disord. 8:S53-S59 (2004); Flynn, “Myobloc,” Dermatol.Clin. 22:207-211 (2004); and Sampaio et al., “Clinical Comparability ofMarketed Formulations of Botulinum Toxin,” Mov. Disord. 8:S129-S136(2004), which are hereby incorporated by reference in their entirety).

A further aspect of the present invention is directed to a method ofdetecting Clostridium botulinum neurotoxin trafficking either formechanistic studies or for imaging specific sites of high neuronalactivity. This method involves expressing a recombinant physiologicallyactive Clostridium botulinum neurotoxin according to the methods of thepresent invention, where a fluorophore is coupled to the neurotoxin, anddetecting trafficking of the neurotoxin by detecting one or morelocations of the fluorophore.

This method of the present invention can be carried out in vivo or invitro.

Detecting location of a fluorophore or other imaging modality can becarried out using methods well-known in the art. For example, detectiondevices are known that are capable of receiving fluorescent emissionsand generating a response to be examined by an operator. Suitabledetectors include, without limitation, charge coupled devices (CCDs),photomultiplier tubes (PMTS), avalanche photodiodes (APDs), andphotodiodes that contain a semiconductor material such as Si, InGaAs,extended InGaAs, Ge, HgCdTe, PbS, PbSe, or GaAs to convert opticalphotons into electrical current. CCD can produce an image in extremelydim light, and its resolution (i.e., sharpness or data density) does notdegrade in low light. Other imaging procedures suitable for detectionmay include CAT scan, PET scan, and MRI.

Another aspect of the present invention relates to a method of detectinglevels of neuronal activity. This method involves providing the isolatedClostridium botulinum neurotoxin of the present invention andadministering the neurotoxin to an individual or a tissue sample. Themethod further involves detecting location of the neurotoxin, wheredetection of the neurotoxin at a specific site in the individual ortissue sample indicates an increased level of activity of neurons atthat site.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1 Creation of the Construct Encoding BoNT/Aad^(ek) in pLitmusVector

Modification of pLitmus28i by Replacement of the Existent Polylinkerwith a Custom-Built Polylinker

A vector with a custom polylinker derived from Litmus 28i (New EnglandBiolabs, Cat. #N3528S, 2823 b.p.) was used for subcloning thefull-length BoNT/Aad^(ek). This derivative of Litmus 28i (pLitmus28C1)was created by restriction digestion of Litmus 28i with Bgl II and AatII followed by dephosphorylation. Annealed phosphorylatedoligonucleotides C1-1S and C1-1A (Table 1) were ligated into digestedLitmus 28i, resulting in the intermediate vector, pLitmus28C1INT (2842b.p.). Vector pLitmus28C1INT was digested with Kas I and Stu I anddephosphorylated. Annealed phosphorylated oligonucleotides C1-2S andC1-2A were ligated into digested pLitmus28C1INT, resulting in the vectorpLitmus28C1 (2890 b.p.). The sequence of this vector was deposited inGenBank under accession number GQ855199, and has a sequence of SEQ IDNO:13, as follows:

gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460ctatagggca gatctgaaga cataagtcgg tccgttcgaa ccagaactct ggaagcttga 2520cgcggccgct atccatggca cacgcgttca gctagcttag gcgcctatgc gcgctaaccg 2580cggtcactta agtatgatat ctctctgcag ttacccgggc atgacgtcta tatgcatatt 2640ctcgaggcat gcgagctccc tcaggaggcc tatagtgagt cgtattacgg actggccgtc 2700gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 2760catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 2820cagttgcgca gcctgaatgg cgaatggcgc ttcgcttggt aataaagccc gcttcggcgg 2880gctttttttt

TABLE 1 Oligonucleotides Used for Cloning Experiments AbbreviationLength Sequence C1-1S 103 b.  5′-pGAT CTG AAG ACA TAA GTC GGT CCG TTCGAA CCA GAA CTC TGG AAG CTT GAC GCG GCC GCT ATC CAT GGC ACA CGC GTT CAGCTA GCT TAG GCG CCT ATG ACG T-3′ (SEQ ID NO: 16) C1-1A 95 b. 5′-pCAT AGGCGC CTA AGC TAG CTG AAC GCG TGT GCC ATG GAT AGC GGC CGC GTC AAG CTT CCAGAG TTC TGG TTC GAA CGG ACC GAC TTA TGT CTT CA-3′(SEQ ID NO: 17) C1-2S108 b.  5′-pGCG CCT ATG CGC GCT AAC CGC GGT CAC TTA AGT ATG ATA TCT CTCTGC AGT TAC CCG GGC ATG ACG TCT ATA TGC ATA TTC TCG AGG CAT GCG AGC TCCCTC AGG AGG-3′(SEQ ID NO: 18) C1-2A 104 b.  5′-pCCT CCT GAG GGA GCT CGCATG CCT CGA GAA TAT GCA TAT AGA CGT CAT GCC CGG GTA ACT GCA GAG AGA TATCAT ACT TAA GTG ACC GCG GTT AGC GCG CAT AG-3′ (SEQ ID NO: 19) CP1S 88 b.5′-pAGC TTA ACT CCT AAA AAA CCG CCA CCA TGA AAT TCT TAG TCA ACG TTG CCCTTG TTT TTA TGG TCG TAT ACA TTT CTT ACA TCT ATG C-3′(SEQ ID NO: 20) CP1A88 b. 5′-pGGC CGC ATA GAT GTA AGA AAT GTA TAC GAC CAT AAA AAC AAG GGCAAC GTT GAC TAA GAA TTT CAT GGT GGC GGT TTT TTA GGA GTT A-3′(SEQ ID NO:21) CP2S 38 b. 5′-pGGC CGC TCA TCA CCA CCA TCA TCA CCA TCA CCA CCACA-3′(SEQ ID NO: 22) CP2A 38 b. 5′-pCGC GTG TGG TGG TGA TGG TGA TGA TGGTGG TGA TGA GC-3′(SEQ ID NO: 23) CP3S 78 b. 5′-pCGC GTG AAA ACC TGT ATTTTC AGG GCG CCG GTG ACT CCC TGT CTT GGC TGC TCC GTC TGC TCA ACG CGC GCGGTG GCG-3′(SEQ ID NO: 24) CP3A 78 b. 5′-pCTA GCG CCA CCG CGC GCG TTG AGCAGA CGG AGC AGC CAA GAC AGG GAG TCA CCG GCG CCC TGA AAA TAC AGG TTTTCA-3′(SEQ ID NO: 25) CP4S 45 b. 5′-pAGC TTA CCA TGG GTC ATC ACC ACC ATCATC ACC ATC ACC ACC ACA-3′(SEQ ID NO: 26) CP4A 45 b. 5′-pCGC GTG TGG TGGTGA TGG TGA TGA TGG TGG TGA TGA CCC ATG GTA-3′(SEQ ID NO: 27) CP5S 45 b.5′-pAGC TTA CGC TGC TCC ATC ACC ACC ATC ATC ACC ATC ACC ACC ACA-3′(SEQID NO: 28) CP5A 45 b. 5′-pCGC GTG TGG TGG TGA TGG TGA TGA TGG TGG TGATGG AGC ATG CTA-3′(SEQ ID NO: 29) CP6 10 b. 5′-pTAC CCT AGG G-3′(SEQ IDNO: 30) CP7S 31 b. 5′-TCA TGC TAG CGT GAG CAA GGG CGC CGA GCT G-3′ (SEQID NO: 31) CP7A 32 b. 5′-TAT AGG TAC CCT TGT ACA GCT CAT CCA TGC CG-3′(SEQ ID NO: 32)

Cloning of BoNT/Aad^(ek) into pLitmus28C1

In the first cloning step, pLitmus28C1 was digested with Hind III andNot I and dephosphorylated. Annealed phosphorylated oligonucleotidesCP1S and CP1A were ligated into digested pLitmus28C1, resulting invector pLitSB3A1 (2968 b.p.). Vector pLitSB3A1 carried several unique5′-restriction sites, including Bgl II, Bbs I, Rsr II, and BstB I, to beused for the subsequent subcloning of the full-length construct intoexpression vectors. This polylinker was followed by the enhancersequence AACTCCTAAAAAACCGCCACC (SEQ ID NO:33), followed by the honeybeemellitin signal peptide sequence.

In the next step, pLitSB3A1 was digested with Not I and Mlu I anddephosphorylated. Annealed phosphorylated oligonucleotides CP2S and CP2Awere ligated into digested pLitSB3A1, resulting in vector pLitSB3A2(2987 b.p.), which carried the sequence encoding a 10-His affinity tagdownstream of the sequence encoding the honeybee mellitin signalpeptide. Next, pLitSB3A2 was digested with Mlu I and Nhe I anddephosphorylated. Annealed phosphorylated oligonucleotides CP3S and CP3Awere ligated into digested pLitSB3A2, resulting in vector pLitSB3A3(3056 b.p.), which carried the sequence encoding the tobacco etch virus(TEV) protease cleavage site, followed by the sequence encoding S6peptide tag downstream of the sequence encoding the 10-His affinity tag.

In the next three steps the synthetic DNA of the full-lengthBoNT/Aad^(ek) with the sequence optimized for expression in two hosts,S. frugiperda and E. coli, was consecutively built into the pLitSB3A3vector. The synthetic gene for full-length BoNT/Aad^(ek) was synthesizedin the form of three contiguous DNA segments, each supplied by thevendor (Genscript) as DNA subcloned in the pUC57 vector. Numbers in thetext below correspond to amino acid numbering in wt BoNT/A. First, a1347 b.p. DNA fragment encoding the sequence P₂-L₄₄₂ was isolated frompUC57-BAad-1 by restriction digest with BssH II and Afl II and subclonedinto pLitSB3A3 (which had been digested with the same set of restrictionenzymes and dephosphorylated) to generate the 4355 b.p. vector,pLitSB3A4. Then, a 1233 b.p. DNA fragment encoding the sequenceD₄₄₃-I₈₄₉ was isolated from pUC57-BAad-2 by digest with Xba I and EcoR Vand subcloned in pLitSB3A4 (digested with the same set of restrictionenzymes and dephosphorylated) to generate the 5570 b.p. vector,pLitSB3A5. Finally, a 1401 b.p. DNA fragment encoding the sequenceP₈₅₀-L₁₂₉₆ followed by a sequence encoding a short linker, the StrepTagII sequence, WSHPQFEK (SEQ ID NO:34), and a triplet of terminationcodons in three reading frames was isolated from pUC57-BAad-3 by digestwith EcoR V and Xho I, and subcloned into pLitSB3A5 (digested with thesame set of restriction enzymes and dephosphorylated) to generate the6925 b.p. vector, pLitSB3A. The sequence of pLitSB3A has been depositedinto GenBank under accession number GQ855200, and has a sequence of SEQID NO:14, as follows:

gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460ctatagggca gatctgaaga cataagtcgg tccgttcgaa ccagaactct ggaagcttaa 2520ctcctaaaaa accgccacca tgaaattctt agtcaacgtt gcccttgttt ttatggtcgt 2580atacatttct tacatctatg cggccgctca tcaccaccat catcaccatc accaccacac 2640gcgtgaaaac ctgtattttc agggcgccgg tgactccctg tcttggctgc tccgtctgct 2700caacgcgcgc ggtggagcta gcggcccgtt cgttaacaaa caatttaact acaaggatcc 2760tgtcaatggt gtggacattg cctatattaa gatcccgaat gcgggtcaga tgcaacccgt 2820gaaagcattc aagatccaca acaaaatctg ggtcatccct gaacgtgaca ctttcacaaa 2880ccctgaagag ggcgacctca accctccccc agaagccaaa caggttccgg tgtcttacta 2940cgatagcacg tacttgtcca ccgataacga gaaggacaac tacctgaagg gagtgaccaa 3000gttgtttgag aggatctact ctaccgatct cggacgtatg ctgctcacga gcattgtgcg 3060cggtatccca ttctggggcg gttcaaccat tgatacagaa ctgaaagtca ttgacactaa 3120ttgtatcaac gttattcaac cagatggcag ctaccgttcc gaggaattga acttggtcat 3180cattggtcca tccgcagaca tcattcagtt tgaatgcaaa tccttcggtc acgaagtgct 3240caacctgacg cgcaacggtt acggctccac ccagtacatc cgtttcagcc ctgatttcac 3300atttggcttc gaggaaagcc tggaggttga caccaacccg ctcctgggtg ctggcaagtt 3360tgcaaccgat cccgcggtga ctctcgctca tgctctgatc cacgccggac accgcctcta 3420tggcatcgct atcaatccga accgcgtgtt caaagtgaat acgaacgcct actatgagat 3480gagcggtctg gaggtttcct ttgaggaact gagaaccttc ggcggtcacg atgccaagtt 3540catcgacagc ttgcaggaaa atgagtttcg cctgtactat tacaacaagt ttaaagacat 3600cgcttccaca ttgaacaaag ccaagtcaat cgtgggtacg acagcttcat tgcagtatat 3660gaagaatgtt ttcaaggaga aatacttgct gtcagaggat acctctggca agttctctgt 3720ggacaaactg aaattcgaca aactgtacaa gatgctgacc gagatttata cggaagataa 3780ctttgtgaaa ttcttcaaag tcctcaacag gaaaactgct ctgaactttg acaaggctgt 3840gttcaagatc aacatcgtcc ccaaagttaa ctacacaatc tatgatggat tcaatctgag 3900aaacaccaac ttggccgcca acttcaacgg ccaaaatacc gaaattaata acatgaattt 3960caccaaactg aagaacttta ctggactgtt cgagttctac aagctgctct gcgtgcgtgg 4020catcatcacc tcacatactc agtctctaga ccagggttat aacgacgatg acgataaagc 4080tctgaacgat ctgtgtatca aggtgaataa ctgggatctg ttctttagcc caagcgagga 4140taacttcacg aacgatctca acaaaggtga agagatcacg tctgatacca atatcgaagc 4200ggctgaagag aatatctcct tggatctcat ccagcaatat tacctgacct ttaacttcga 4260taacgagccc gaaaacatct ccatcgagaa cctcagctca gacatcattg gtcagttgga 4320gctgatgcca aacattgaac gcttccccaa cggcaagaaa tacgaactcg acaagtatac 4380gatgtttcat tacttaagag cgcaggagtt tgaacacggc aagagccgca ttgctctcac 4440taactccgtg aatgaagccc tgctcaatcc gtcaagggtg tacacattct ttagctccga 4500ctatgtcaag aaagtgaaca aagccaccga agcggcaatg ttcctgggat gggttgaaca 4560actggtctac gacttcaccg acgagacctc tgaggtgagc acaacggaca agattgctga 4620catcactatc attatcccgt atattggacc tgccttgaat attggcaaca tgctctacaa 4680agacgatttc gttggtgccc tgatcttcag cggtgccgtg atcctgttgg agttcattcc 4740tgaaatcgcc atccctgtgc tgggcacgtt cgctctggtc tcatacattg cgaataaggt 4800cttgaccgtg cagacaatcg ataatgccct ctccaaacgt aacgaaaaat gggacgaggt 4860ctacaaatac atcgtgacca actggctggc aaaggttaac acccaaattg atctgatccg 4920taagaaaatg aaggaggctt tggagaacca ggctgaagct actaaagcca ttatcaacta 4980ccagtataat cagtatacag aagaggaaaa gaataacatc aatttcaaca tcgatgactt 5040gtcctcaaag ctgaacgagt ccatcaacaa agctatgatc aacatcaaca aattcctgaa 5100tcagtgctcc gtgtcttacc tgatgaactc tatgatccca tacggtgtga agcgcctgga 5160ggacttcgat gccagcctga aagacgcact gctcaaatac atttacgata atcgcggcac 5220tttgattggc caagttgacc gtctgaagga caaggttaac aataccttgt caaccgatat 5280cccctttcaa ctgtccaaat acgttgataa ccagcgcttg ctctctactt tcaccgaata 5340cattaacaac attatcaata catcaattct caacctgcgc tatgagtcca atcatctgat 5400cgatctgtct cgttacgcca gcaagatcaa cattggcagc aaagtgaact tcgatccgat 5460tgacaagaac caaatccagt tgttcaacct cgaaagctcc aaaatcgaag tgatcctgaa 5520gaatgccatc gtctacaact ccatgtatga aaatttctca acttcattct ggattagaat 5580cccgaaatac ttcaactcaa tctctctgaa taacgaatac acgatcatta actgtatgga 5640gaataactct ggttggaagg tttccttgaa ctatggagaa attatctgga ctctgcaaga 5700tacgcaagag atcaaacagc gtgtggtctt taaatacagc cagatgatta acatctctga 5760ctacatcaac agatggatct ttgtcaccat tacaaacaat cgcctgaata actccaaaat 5820ctacatcaac ggtcgtctga tcgaccagaa acctatttca aacctcggca acattcatgc 5880ttccaataac atcatgttta agttggatgg ttgccgcgat acccaccgtt acatctggat 5940caagtatttc aatctgttcg acaaagaact caatgagaaa gagatcaaag acttgtatga 6000taatcagtca aactccggca ttctgaaaga cttctggggc gattacctcc agtacgataa 6060gccatattac atgctgaatc tctatgaccc taacaaatat gtggacgtga acaatgtcgg 6120tatccgtggc tacatgtacc tcaaaggacc acgtggtagc gttatgacaa ccaacatcta 6180cctgaatagc tccttgtatc gcggtacgaa gttcattatc aagaagtacg cttcaggcaa 6240caaggacaac atcgtgagga acaatgatcg cgtgtacatc aacgtcgtgg tgaagaataa 6300ggaataccgc ttggcgacca acgcttctca ggctggagtt gagaagatcc tgagcgcctt 6360ggagatccca gacgttggca acctgagcca agtggttgtg atgaaaagca agaatgacca 6420gggaatcacc aacaaatgca aaatgaacct gcaagacaac aacggcaatg acatcggttt 6480catcggtttc caccagttta acaatattgc gaagctggtc gccagcaact ggtacaacag 6540gcagattgag aggtcatccc gtaccttagg atgctcttgg gaatttatcc ccgtggacga 6600tggttggggc gagagacccc tgggcgcagg ttggtcccac cctcagttcg agaagtaata 6660gttaatagat aataatagct cgaggcatgc gagctccctc aggaggatag tgagtcgtat 6720tacggactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 6780aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 6840gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcttcgc ttggtaataa 6900agcccgcttc ggcgggcttt ttttt

Example 2 Creation of Vectors for Protein Expression in E. coli

pET-19b-Based Construct

Vector pLitSB3A was linearized by digestion with Hind III and Mlu I anddephosporylated. The 6799 b.p. DNA fragment was isolated from an agarosegel and purified. Synthetic phosphorylated oligonucleotides CP4S andCP4A were annealed and ligated into the isolated vector DNA to generatethe vector pLitB3A1 (6844 b.p.). Vector pLitB3A1 was digested with Nco Iand Xho I and the 4078 b.p. DNA fragment was isolated and purified froman agarose gel. This fragment was ligated into expression vector pET-19b(Novagen, Cat. #69677, 5717 b.p.), that had been digested with Nco I andXho I and dephosphorylated, generating the 9721 b.p. vector pET19B3A.

pETcoco2-Based Construct

Vector pLitSB3A was linearized by digestion with Hind III and Mlu I anddephosphorylated. The 6799 b.p. DNA fragment was isolated from anagarose gel and purified. Synthetic phosphorylated oligonucleotides CP5Sand CP5A were annealed and ligated into the isolated vector DNA togenerate the vector pLitB3A2 (6844 b.p.). Vector pLitB3A2 was digestedwith Xho I and treated with CpG methyltransferase (M. SssI, New EnglandBiolabs, Cat. #M0226). After completion of methylation, the enzyme wasinactivated and removed by phenol/chloroform extraction, followed by DNAprecipitation with ethanol. DNA was treated with Klenow fragment of DNApolymerase I (New England Biolabs, Cat. #M021) to fill in 5′ overhangsgenerated by Xho I cleavage, and ligated. Ligated DNA was digested withPvu I and dephosphorylated. Annealed phosphorylated syntheticoligonucleotide CP 6 was ligated into Pvu I digested vector to generatepLitB3A3 (6858 b.p.). This vector was transformed, amplified, andpropagated in NEB 10-beta E. coli strain (New England Biolabs, Cat.#C3020K). Following propagation, plasmid DNA was isolated and digestedwith restriction endonucleases Sph I and Avr II. The 4081 b.p. DNAfragment was isolated from an agarose gel, purified, and ligated intothe vector pETcoco2 (Novagen, Cat. #71148-3, 12417 b.p.) (that had beentreated with the same set of restriction endonucleases anddephosphorylated) to generate the expression vector pETcocoB3A (16263b.p.).

Example 3 Creation of Vectors for Protein Expression in Baculovirus

Construct for Expression of BoNT/Aad^(ek)

The plasmid pLitSB3A was digested with the restriction endonucleases RsrII and Xho I. The 4190 b.p. DNA fragment, encoding full-lengthBoNT/Aad^(ek), was isolated from an agarose gel, purified, and ligatedinto the baculovirus transposition vector, pFastBac1™ (Invitrogen, Cat.#10360-014, 4776 b.p.) (which had been digested with Rsr II and XhoI anddephosphorylated) to generate the 8968 b.p. vector pFB1SB3AEK. The DNAsequence of pFB1SB3AEK with annotations has been deposited to GenBankunder accession number GQ855201 (SEQ ID NO:15). Features of the 1372 aaprotein, translated from the open reading frame of the insert DNA(BoNT/Aad^(ek)), are shown in FIG. 1.

Construct for Expression of BoNT/Aad^(tev)

In this construct, DNA encoding the enterokinase cleavage sitepositioned between LC and HC was replaced with a sequence encoding asecond TEV cleavage site. The 348 b.p. fragment containing theenterokinase cleavage site was excised from pFB1SB3AEK by digestion withXba I and Afl II. The linearized vector was dephosphorylated and a 351b.p. Xba I/Afl II DNA fragment excised from the plasmid pUC57-TEV(GenScript) was ligated into the vector to generate the 8971 b.p. vectorpFB1SB3ATEV. The DNA sequence of pFB1SB3ATEV, along with annotations,has been deposited to GenBank under accession number GQ855202 (SEQ IDNO:10). Features of the 1373 aa protein, translated from the openreading frame of the insert DNA (BoNT/Aad^(tev)), are shown in FIG. 1.

Construct for Expression of ΔLC-Peptide-BoNT/A^(tev)

Vector pFB1SB3ATEV was linearized by digestion with BssH II and Xba Iand dephosphorylated. The 7631 b.p. dephosphorylated vector was isolatedfrom an agarose gel and purified. A DNA fragment containing the sequenceencoding the ΔLC-peptide was obtained in the form of synthetic DNAcloned into pUC57 (pUC57-Pept), supplied by the manufacturer(GenScript). pUC 57-Pept was digested with BssH II and Xba I and the 200b.p. DNA fragment was isolated from an agarose gel, purified, andligated into the linearized pFB1SB3ATEV, resulting in the 7831 b.p.vector, pFB1SPepB2ATEV. The DNA sequence of pFB1SPepB2ATEV, along withannotations, have been deposited in GenBank under accession numberGQ855203 (SEQ ID NO:11). Features of the 993 aa protein, translated fromthe open reading frame of the insert DNA (ΔLC-Peptide-BoNT/A^(tev)), areshown in FIG. 1.

Construct for Expression of ΔLC-GFP-BoNT/A^(tev)

DNA encoding the GFP sequence was obtained by PCR amplification fromplasmid pAcGFP1-C2 (Clontech, Cat. #632481, 4722 b.p.) using primersCP7S and CP7A (Table 1) and PrimeSTAR HS DNA polymerase (Takara, Cat.#TAK R010A) in a 50 μl reaction mixture in a GeneAmp PCR system 9700(PE/Applied Biosystems). The reaction buffer and conditions were setaccording to the protocol provided by Takara. The PCR product wasdigested with restriction endonucleases Nhe I and Acc65 I, and the 700b.p. digested DNA was isolated from an agarose gel and purified. VectorpFB1SPepB2ATEV was digested with Nhe I and under digested (shortincubation time and small amount of the restriction endonuclease) withAcc65 I. The 7822 b.p. DNA fragment was isolated from an agarose gel,purified, and dephosphorylated. The purified GFP PCR product and thelinearized pFB1SPepB2ATEV were ligated, resulting in the 7822 b.p.vector pFB1SGFPB2ATEV. The DNA sequence of pFB1SGFPB2ATEV, along withannotations, has been deposited to GenBank under accession numberGQ855204 (SEQ ID NO:12). Features of the 1230 aa protein, translatedfrom the open reading frame of the insert DNA (ΔLC-GFP-BoNT/A^(tev)),are shown in FIG. 1.

Custom synthetic oligonucleotides were obtained from Sigma-Aldrich. E.coli strain TOP10, used for plasmid transformation and amplification,was purchased in a form of electrocompetent cells from Invitrogen (Cat.#C404052), except as specified otherwise. Restriction endonucleases, T4DNA ligase, and arctic shrimp alkaline phosphatase were purchased fromNew England Biolabs. All DNA fragments isolated from agarose gels afterenzymatic treatment were purified with Qiaex II DNA extraction kit(Qiagen, Cat. #20051). DNA sequences of all final constructs wereobtained using overlapping sets of primers at the DNA sequencingfacility at NYU's Skirball Institute of Biomolecular Medicine. Thesequencing data obtained proved constructs to be free of unexpectedmutations.

Example 4 Protein Expression and Purification—E. coli Experiments

Electrocompetent E. coli strain JM109 (DE3) (Promega, Cat. #P9801) wastransformed with pET19B3A plasmid according to an established protocol.A single colony isolated from a Luria-Bertani (LB) plate containingcarbenicillin (100 μg/ml) was inoculated into 10 ml of LB mediumcontaining carbenicillin and grown overnight with agitation (250 rpm) at37° C. An aliquot (2 ml) of the culture was transferred to 200 ml offresh LB medium with the addition of carbenicillin (100 μg/ml), and thecells were grown further at 37° C. with agitation to reach an OD₆₀₀ ofapproximately 0.5 (about 4 hours). The suspension was rapidly cooled toapproximately 25° C. in an ice bath, and isopropyl-β-D-thiogalactoside(IPTG) was added to a final concentration of 1 mM. Induction was allowedto proceed for 12 hours at 25° C. Aliquots were collected at 3, 6, and12 hours after IPTG induction and cells were harvested by centrifugationat 6000 g for 15 min at 4° C. The cells were resuspended at 4° C. inBugBuster protein extraction reagent (Novagen, Cat. #70584) and lysedwith the addition of Lysonase™ bioprocessing reagent (lysozyme andbenzonase, Novagen, Cat. #71230-4) in the presence of protease inhibitorcocktail without EDTA (Pierce, Cat. #78430) by triturating thesuspension until signs of high viscosity disappeared (approximately 30minutes), The BugBuster lysate was cleared by centrifugation at 35,000 gfor 15 minutes, 4° C., distributed in 0.5 ml aliquots and stored at −80°C. The residual pellet was resuspended at room temperature in 6 M urea,25 mM Tris-HCl, pH 8.0. Cleared urea solubilizate was collected bycentrifugation at 35,000 g for 15 minutes at room temperature,distributed in 0.5 ml aliquots, and stored at −80° C. Aliquots ofBugBuster and urea lysates were batch incubated with TALON® affinityresin (Clontech, Cat. #63506) at 4° C. for 30 minutes. TALON® beads weresequentially washed with 100 mM NaCl, 25 mM Tris HCl-pH8.0 and proteinwas eluted by resuspension of the beads in 1× SDS PAGE loading bufferwith β-mercaptoethanol (Bio-Rad, Cat. #161-0710). BugBuster and urealysates, as well as eluates of these lysates obtained from Talon® resin,were loaded on a 10.5-14% Criterion SDS PAGE gel (Bio-Rad, Cat.#345-0106) and separated. Proteins were transferred to nitrocellulosemembranes (Bio-Rad, Cat. #162-0117) and probed with polyclonalantibodies against BoNT/A holotoxin (Pol001, raised against BoNT/Aholotoxoid (Staten Serum Institut, Denmark)). As a positive control, mBoNT/A was loaded on the gel, and JM109 (DE3) transformed with the emptyvector was used as the negative control. When the results were analyzedby elecrophoresis and Western blotting, it was unexpectedly found thattoxin of the proper molecular weight and immunoreactivity could not beproduced.

pETcocoB3A was transformed into competent E. coli Rosetta-gami B (DE3)cells (Novagen, Cat. #71136-3) according to the manufacturer's protocol.A single bacterial colony, picked from an LB agar plate, was grown in(i) LB medium with 100 μg/ml carbenicillin, 15 μg/ml kanamycin, 12 μg/mltetracycline, and 34 μg/ml chloramphenicol, and (ii) LB medium with0.01% L-arabinose, 100 μg/ml carbenicillin, 15 μg/ml kanamycin, 12 μg/mltetracycline, and 34 μg/ml chloramphenicol for 16 hours at 37° C. Analiquot of overnight cultures (1:100 v/v) were transferred to fresh LBmedia supplemented with antibiotics, with and without addition ofL-arabinose. The cells were grown at 37° C. with agitation to reachOD₆₀₀ of approximately 0.4 (about 9 hours). The suspensions were rapidlycooled to approximately 25° C. in an ice bath, and IPTG was added to afinal concentration of 0.5 mM. The cultures were incubated withagitation for 12 hours at 25° C. Aliquots were collected at 3, 6, and 12hours after IPTG induction and cells were harvested by centrifugation at6000 g for 15 min at 4° C. Protein extraction, sample preparation, SDSPAGE, and Western blotting were performed as described above. When theresults were analyzed by elecrophoresis and Western blotting, it wasunexpectedly found that toxin of the proper molecular weight andimmunoreactivity could not be produced.

Example 5 Baculovirus Experiments

Generating Recombinant Bacmids

pFastBac™ constructs were used for transposition of the cloned DNA intothe shuttle vector (bacmid). Approximately 1 ng of plasmid DNA was usedto transform MAX Efficiency® DH10Bac™ E. coli (Invitrogen, Cat.#10361-012) using a heat-shock method according to the manufacturer'sprotocol (Invitrogen, BactoBac® baculovirus expression system). Colonieswere grown on LB agar plates supplemented with 50 μg/ml kanamycin, 7μg/ml gentamycin, 10 μg/ml tetracycline, 100 μg/ml Bluo-Gal, and 40μg/ml IPTG, for 48 hours for full color development. For each construct,two white colonies were picked and re-grown for 24 hours at 37° C. in 50ml LB medium supplemented with antibiotics. The recombinant bacmids wereisolated and purified from harvested cells using a QIAGENLarge-Construct Kit (Cat. #12462), according to the manufacturer'sprotocol. The DNA concentration was measured with a NanoDropspectrophotometer and adjusted to 0.5 mg/ml with TE buffer. The yield ofthe bacmid DNA was approximately 1 μg/ml of starting LB culture.Transposition of the cloned genes into recombinant bacmids was confirmedby PCR with bacmid as a template and sets of primers specific for eachcloned construct.

Example 6 Transfecting Insect Cells and Collecting Baculoviral Stock forProtein Expression

Sf9 insect cells, grown in shaker flasks in serum-free Sf-900 II medium(Invitrogen, Cat. #10902-096) were plated in 6-well (35 mm) cultureplates at a density of 1×10⁶ cells per well. Plates were incubated at28° C. in a humidified incubator until cells adhered to the surface(approximately 1 hour). Two (2) μg of bacmid DNA (0.5 mg/ml) was dilutedwith 100 μl unsupplemented Grace's insect cell medium (Invitrogen, Cat.#11595-030) in a 15-ml sterile polystyrene tube. In a separate tube, 7μl of CELLFECTIN® reagent (Invitrogen, Cat. #10362-010) was also dilutedwith 100 μl of unsupplemented Grace's medium. Diluted bacmid DNA andCELLFECTN® were combined and incubated at room temperature for 40minutes. While DNA:lipid complexes were incubating, the medium wasaspirated from the adherent Sf9 cells, cells were washed withunsupplemented Grace's medium, and 2 ml of fresh Grace's medium wasadded to each well. At the end of the 40 minute incubation, 800 μl ofunsupplemented Grace's medium was added to the DNA-CELLFECTIN® mixture,the medium covering the cells was aspirated, and 1 ml of theDNA-CELLFECTIN® mixture was gently added to the adherent Sf9 cells.Cells were incubated at 28° C. for 5 hours, after which the DNA-lipidcomplexes were aspirated and 2 ml of fresh Sf-900 II serum-free mediumwas added to the cells and incubation was continued. P1 low-titerbaculoviral stock (2 ml of growth medium) was collected 72 hours aftertransfection.

To generate P2 high-titer stock, 1 ml of P1 stock was added to 25 ml ofSf9 grown in Sf-900 II medium in a shaker flask. Cell density at thetime of infection was approximately 1.5×10⁶ cells/ml. Cells wereincubated for 72 hours at 28° C. in a humidified incubator. Cells wereremoved from high titer P2 stock by centrifugation at 1000 g. The titerof the P2 stock was measured by viral plaque assay (Invitrogen,BAC-TOBAC® baculovirus expression system). P2 titers for the constructswere approximately 1-2×10⁸ pfu/ml. P2 stock was used to infect Sf9 cellsfor expression of the proteins of interest.

Example 7 Protein Expression, Purification, and Processing

Sf9 cells grown to a density of approximately 1.5×10⁶ cells/ml in ashaker flask in Sf-900 II serum-free medium were infected withrecombinant P2 baculovirus stock. For each recombinantprotein-expression vector, the optimal multiplicity of infection (MOI)and time after infection for harvesting was determined empirically. ForBoNT/Aad^(ek) and BoNT/Aad^(tev), these conditions were MOIapproximately 0.1 and 60 hours; for ΔLC-Peptide-BoNT/A^(tev) and ΔLC-GFP-BoNT/A^(tev), the conditions were MOI approximately 0.5 and 72 hours.At the time of harvest, medium was separated from the cells bycentrifugation at 1000 g, filtered through a Whatman GF/F filter(Schleicher&Schuell, Cat. #1825090), concentrated approximately 3 foldon a Millipore Prep/Scale TFF cartridge (Cat. #CDUF002LT), and dialyzedagainst TALON® resin chromatography loading buffer (500 mM NaCl, 25 mMTrisHCl, 5 mM imidazole, pH 8.0). For ΔLC-Peptide-BoNT/A^(tev) and ΔLC-GFP-BoNT/A^(tev), 0.3% Triton X-100 was added to the medium prior toconcentration. Dialyzed supernatant was loaded on TALON® resin(Clontech, Cat. #63506) pre-equilibrated with the loading buffer. Afterloading, resin was washed with 10 volumes of loading buffer, 20 volumesof wash buffer (500 mM NaCl, 25 mM TrisHCl, 20 mM imidazole, pH 8.0),and protein was eluted with 5 volumes of elution buffer (500 mM NaCl, 25mM TrisHCl, 200 mM imidazole, pH 8.0). For ΔLC -Peptide-BoNT/A^(tev) andΔLC-GFP-BoNT/A^(tev) derivatives, chromatography was slightly modifiedby inclusion of 1% TRITON® X-100 in the loading buffer. For thesederivatives, after loading and column wash with 10 volumes of loadingbuffer, the column was washed with 10 volumes of 20 mM imidazole washbuffer with 1% TRITON® X-100 followed by washing with 10 volumes of 20mM imidazole wash buffer without detergent. Protein was eluted with 5volumes of elution buffer containing 0.2% n-octyl-β-D-glucopyranoside(USB, Cat. #29836-26-8). Eluted proteins were dialyzed against 100 mMNaCl, 25 mM TrisHCl buffer, pH 8.0, either without (BoNT/Aad^(ek) andBoNT/Aad^(tev)), or with (ΔLC-Peptide-BoNT/A^(tev) and ΔLC-GFP-BoNT/A^(tev)) 0.2% n-octyl-β-D-glucopyranoside, and concentratedusing Millipore Amicon Ultra 30,000 MWCO centrifugal filter units (Cat.#UFC803024) to a final total protein concentration of approximately 10mg/ml. Dialyzed and concentrated fractions were loaded on StrepTactinsuperflow agarose (Novagen, Cat. #71592) pre-equlibrated with 100 mMNaCl, 25 mM TrisHCl buffer, pH 8.0 with or without 0.2%n-octyl-β-D-glucopyranoside, and washed with the same buffer. Boundprotein was eluted with 3 mM D-desthiobiotin (Novagen, Cat. #71610).Fractions containing pure protein were combined, dialyzed, andconcentrated using Amicon Ultra centrifugal filter units.

Processing of BoNT/Aad^(ek)

The 10-His tag was removed from recombinant protein using AcTEV protease(Invitrogen, Cat. #12575-015). The protein was incubated with AcTEVprotease (1 unit of enzyme per 1 μg of protein) at 30° C. for 6 hours indigestion buffer (50 mM NaCl, 0.5 mM EDTA, 3 mM glutathione, 0.3 mMoxidized glutathione, pH 8.0). Completion of the cleavage was confirmedby Western blotting with anti-His monoclonal antibodies (Santa Cruz, H-3His probe, Cat #sc-8036 HRP). The AcTEV and potentially under-digestedprotein were removed from the reaction mixture by TALON RESIN®chromatography. De-tagged protein was collected in the flow-throughfractions, which were analyzed, combined, dialyzed against low-saltbuffer, and concentrated using Amicon Ultra centrifugal filter units.Propeptide was converted to the heterodimer by treatment withrecombinant enterokinase (rEK, Novagen, Cat. #71537-3). The protein wasincubated with Tag-off high activity rEK (0.007 units of enzyme per 1 μgof the protein) at 16° C. for 16 hours in digestion buffer (50 mM NaCl,20 mM TrisHCl, 2 mM CaCl₂, pH 7.8). Approximately 95% of the protein wasprocessed to heterodimer by this treatment as assessed byCoomassie-stained SDS-PAGE under reducing conditions. The rEK wasremoved from the reaction mixture by Tag-off cleavage capture kit(Novagen, Cat. #71540-3), according to the manufacturer's protocol.Protein was dialyzed against buffer (100 mM NaCl, 40 mM sodiumphosphate, 40% glycerol, pH 7.2) and concentrated using Amicon Ultrafiltration units.

Processing of BoNT/A ad^(tel), ΔLC-Peptide-BoNT/A^(tev), andΔLC-GFP-BoNT/A^(tev)

N-terminally placed 10-His tag removal and propeptide cleavage wereperformed simultaneously, according to the protocol described forBoNT/Aac^(ek) above. ΔLC-Peptide-BoNT/A^(tev) and ΔLC-GFP-BoNT/A^(tev)required the presence of 0.2% n-octyl-β-D-glucopyranoside during thecleavage. The detergent was removed after cleavage by dialysis andconcentration on Amicon Ultra filtration units.

Example 8 Enzymatic Labeling of BoNT/Aad^(ek)

All expressed proteins incorporated S6 peptide GDSLSWLLRLLN (SEQ IDNO:2) (Zhou et al., “Genetically Encoded Short Peptide Tags forOrthogonal Protein Labeling by Sfp and AcpS PhosphopantetheinylTransferases,” ACS Chem. Biol. 2(5):337-346 (2007), which is herebyincorporated by reference in its entirety) at the N-terminus of thesmaller subunit of the heterodimer The pET29 vector with clonedC-terminally His6-tagged Sfp phosphopantetheinyl transferase from B.subtilis was kindly provided by Dr. Jun Yin (University of Chicago).Recombinant Sfp phosphopantetheinyl transferase was expressed in E. colistrain BL21 (DE3) (Novagen, Cat. #69450) and purified according to theprotocol. CoA 547 was purchased from Covalys (Cat. #SV124) or from NewEngland Biolabs (Cat. #S9349S). Prior to enzymatic labeling, thefluorescent substrate was dissolved in DMSO to a final concentration of1 mM. Aliquots of this solution were used as a stock for furtherdilution in aqueous buffer-based labeling preparations. The labelingreaction was optimized and performed according to the followingprotocol: The molar ratio of labeled protein: Sfp phosphopantetheinyltransferase: CoA 547 was 4:1:5. A pilot reaction was performed byassembling approximately 18 μg of processed BoNT/Aad^(ek) in a 30 μlreaction mixture containing 1× unsupplemented Grace's insect cellculture medium (prepared from the dry medium, Invitrogen, Cat.#11300-043), 5 mM MgCl₂, mixture of short peptides used to suppress thebackground labeling and consisting of 150 μM Neurokinin A (PeninsulaLaboratories, Cat. #7359), 75 mM Substance P (Peninsula Laboratories,Cat. #7451), 2 mM poly-L -lysine hydrobromide (MW 500-2000) (Sigma, Cat.#P8954), 1 μM Sfp phosphopantetheinyl transferase and 5 μM CoA 547, pH7.0. Fluorescent substrate was added as the final component and thereaction mixture was incubated for 15 minutes at 30° C. Forvisualization after labeling, the reaction was stopped by the additionof 2× Laemmli SDS loading buffer followed by SDS PAGE, transfer of theproteins to a nitrocellulose membrane, and scanning on a Typhoonscanner. For the preparative isolation of labeled proteins, after the 15minute incubation, 10 volumes of TALON® resin loading buffer (500 mMNaCl, 25 mM TrisHCl, pH 8.0) was added to the reaction mix and theresulting solution was passed through TALON® chelating resin. Flowthrough fractions and three additional washes (each corresponding to thecolumn volume) were combined and immediately concentrated using Amiconultrafiltration units. Concentrated protein was dialyzed againstglycerol-phosphate buffer (100 mM NaCl, 40 mM sodium phosphate, 40%glycerol, pH 7.2). All procedures that involved fluorescent substrate orlabeled protein were performed in the dark.

Example 9 Structural Analysis of BoNT/Aad^(ek) and Fluorescently LabeledBoNT/Aad^(ek)

Edman Degradation

BoNT/Aad^(ek) LC and HC, separated under reducing conditions on 10-14%Criterion SDS PAGE (Bio-Rad), were transferred to a PVDF membrane(Bio-Rad, Cat. #162-0182) and stained with Bio-Safe Coomassie BB G-250(Bio-Rad, Cat. #161-0786). Proteins were subjected to sequencing on anABI 494-HT Procise Edman Sequencer at the Molecular Structure Facilityat UC Davis.

In-Gel Tryptic Digestion

Gel bands were destained until clear using 30% acetonitrile in 25 mMammonium bicarbonate. The gel slices were then cut into smaller pieces,approximately 1 mm³ in size, and placed in a Speed Vac vacuum centrifugefor complete dehydration. After dehydration, 25 μl of 0.1% RapiGest SF(Waters, Milford, Mass.) was added and the samples were incubated at 37°C. for 10 min. All remaining supernatant fluid was discarded and thesamples were placed in a Speed Vac vacuum centrifuge for completedehydration. Then 10 ng/μl sequencing grade trypsin (PromegaCorporation, Madison, Wis.) in 50 mM ammonium bicarbonate was added toeach sample and they were left to digest overnight at 37° C. Sampleswere extracted stepwise using acetonitrile, 1% formic acid, andacetonitrile again. Samples were vortexed at 800 rpm between steps andsupernatant fluids were transferred to 0.5 ml Eppendorf tubes. Then 10μl of 10% TFA was added to each extract followed by incubation at 37° C.for 10 min. Samples were then dried in the vacuum centrifuge untilapproximately 3 μl of extract remained.

MALDI-TOF Analysis of BoNT/Aad^(ek) Tryptic Digest

Samples were desalted using C18 ZipTips (Millipore, Billerica, Mass.)and eluted with 50% acetonitrile, 0.1% formic acid after which 1 μl ofsample was combined with 1 μl of alpha-cyano-4-hydroxycinnamic acid(Agilent Technologies, Santa Clara, Calif.). Sample (1 μl) was thenspotted on a Bruker 384 steel target frame (Billerica, Mass.) andallowed to air dry at room temperature. Samples were analyzed using aBruker Autoflex MALDI-TOF mass spectrometer in positive ion reflectronmode under standard operating conditions.

HPLC-Q-TOF MS/MS Analysis

A Q-TOF Premier mass spectrometer (Waters, Milford, Mass.) equipped witha Waters nano-ESI source coupled directly to a Nano-Acquity UPLC system(Waters) equipped with a 100 μm×15 cm reverse phase column (BEH C18,Waters) was used for all LC-MS/MS analyses. Samples were directly loadedon the column using 0.1% formic acid at a flow rate of 0.8 μl/min for 20min and eluted by a gradient of 1-40% acetonitrile in 0.1% formic acidover 40 min.

Samples for quantitation were analyzed by the mass spectrometer inTOF-MS mode while samples for identification by MS/MS were analyzed inDDA (data-dependent acquisition) mode. The mass range for all surveyscans was 300-1400 m/z. Mascot software (version 2.2.1, Matrix Science,London, U.K.) was used for database searching and spectralinterpretation.

Example 10 Expression, Purification, and Processing of BoNT/Aad^(ek)

The full-length BoNT/A ad (atoxic derivatives) DNA and proteins weregenerated under biosafety level 2 containment (project approved by CDCon Feb. 7, 2006 for the registered entity C20060207-0419).

To improve the yield of the recombinant protein, the DNA sequenceencoding the full-length construct was optimized for expression in bothand Sf9 insect cells and E. coli and synthesized de novo, as explainedin supra.

A four amino acid insert, DDDD (SEQ ID NO:44), was introduced betweenamino acid residues N₄₄₇ and K₄₄₈ in the first construct, BoNT/Aad^(ek),to create a site recognized and cleaved by enterokinase (FIG. 1).

E. coli Experiments

The BoNT/Aad^(ek) insert was initially created in a non-expressionvector derived from the Litmus 28i plasmid and named pLitSB3A. Theinsert DNA contains a 5′ enhancer sequence placed upstream of theinitiation codon and signal peptide, both of which are used exclusivelyfor protein expression in the baculovirus system. To generate a vectorsuitable for E. coli expression, the DNA sequence encoding full-lengthBoNT/Aad^(ek) was isolated from pLitSB3A and subcloned downstream of theT7lac promoter into expression vector pET 19. The resulting vector wastransformed into E. coli strain JM109 (DE3), followed by growth of thetransformed culture, IPTG induction, harvesting, lysis, SDS PAGE, andWestern blotting with BoNT/A polyclonal antibodies. Under the conditionstested, BoNT/A-specific immunoreactivity in the processed samples wasunable to be detected.

Next, expression of BoNT/Aad^(ek) was tested in a different expressionvector and different E. coli strain. DNA encoding full-lengthBoNT/Aad^(ek) was isolated from pLitSB3A and subcloned downstream ofT7lac promoter in the expression vector pETcoco2. The pETcoco systemcombines the advantages of T7 promoter-driven protein expression withthe ability to control plasmid copy number. The pETcoco vectors arenormally maintained at one copy per cell. In the single-copy statepETcoco clones are extremely stable, which is especially important fortarget genes toxic to the host. Copy number can be amplified to 20-50copies per cell by the addition of L-arabinose to the culture medium. InλDE3 lysogenic hosts carrying pETcoco vectors expression of the targetgene can be increased by as much as 2,500-fold over background when IPTGis added to the culture media. The expression construct was transformedinto E. coli Rosetta-gami B (DE3) competent cells and was grown in LBmedia with carbenicillin, kanamycin, tetracycline, and chloramphenicol.Carbenicillin was added to maintain propagation of the cells carryingthe pETcoco-based bla marker, kanamycin and tetracycline were added toselect for thioredoxin (irxB) and glutathione reductase (gor) mutations,thus improving the chances for proper disulfide bond formation in the E.coil cytoplasm (Derman et al., “Mutations that Allow Disulfide BondFormation In the Cytoplasm of Escherichia coli,” Science262(5140):1744-1747 (1993); Prinz et al., “The Role of the Thioredoxinand Glutaredoxin Pathways in Reducing Protein Disulfide Bonds In theEscherichia coli Cytoplasm,” J. Biol. Chem. 272(25):15661-15667 (1997),which are hereby incorporated by reference in their entirety).Chloramphenicol was added to the medium to maintain helper plasmids thatprovide tRNAs for rare codons, thereby increasing the expression ofproteins encoded by DNA with codons non-canonical for E. coli. Multipleconditions were tested to optimize expression of the BoNT/Aad^(ek).Cultures were grown with and without L-arabinose in the media, anddifferent IPTG concentrations were evaluated for induction. Incubationtemperatures and times were also optimized for expression. Optimalconditions were as follows: E. coli cultures were incubated overnight inthe presence of L-arabinose at 37° C. until reaching an OD₆₀₀ ofapproximately 0.4. The temperature of the bacterial suspension was thenlowered to 25° C. over 30 minutes, and IPTG was added to a finalconcentration 0.5 mM. After induction, culture growth was allowed tocontinue on a shaker incubator at 25° C. for twelve more hours. Thebacterial pellet was harvested by centrifugation, lysed with BugBusterlysis reagent in the presence of benzonase, lysozyme, and a cocktail ofprotease inhibitors, The lysate was cleared by centrifugation andpurified by incubation with a TALON® affinity resin. The supernatant andeluate from the TALON® beads were run on SDS PAGE and analyzed byWestern blot with polyclonal antibodies raised against the full-lengthBoNT/A holotoxin. Rosetta-gami B (DE3), transformed with the emptyvector was used as the negative control. The expressed protein waspartially soluble, immunoreactive to the polyclonal BoNT/A antibodies,and could be purified using the metal affinity tag. However, themolecular weight of the recombinant propeptide expressed wassignificantly lower than that of the native full-length BoNT/Apropeptide. Extensive proteolysis was observed with all purification andexpression protocols tested, even when the derivative was expressed withthe single-copy plasmid. This instability may be related to the systemsavailable in E. coli for post-translational processing of proteins, withimproper folding and disulfide bonding making the recombinant proteinsusceptible to degradation.

Baculovirus Expression

The insert for expression of BoNT/Aadek was isolated from pLitSB3A andsubcloned into the donor vector pFastBacl under control of thepolyhedrin promoter. A 21 b.p. cis-DNA sequence AACTCCTAAAAAACCGCCACC(SEQ ID NO:35) that was shown to increase the expression levels ofexogenous genes in baculovirus-infected insect cells (Sano et al.,“Enhancement of Protein Expression In Insect Cells by a LobsterTropomyosin cDNA Leader Sequence,” FEBS Lett. 532(1-2):143-146 (2002),which is hereby incorporated by reference in its entirety) waspositioned in front of the first methionine, upstream of DNA encodingthe honeybee mellitin signal peptide MKFLVNVALVFMVVYISYIYAA (SEQ IDNO:36). The signal peptide is needed for transport of the expressedprotein into the culture medium and is removed by processing duringintracellular trafficking and secretion (von Heijne, “Signals forProtein Targeting Into and Across Membranes,” Subcell. Biochem. 22:1-19(1994), which is hereby incorporated by reference in its entirety). Forthe purpose of protein purification, an N-terminal 10-His tag andC-terminal Strep-tag II were also present in the construct. Thegeneration of recombinant baculovirus and insect cell procedures aredetailed supra. The expressed propeptide was detected in the secretedmedium with polyclonal antibodies raised against BoNT/A holotoxin. Themobility of the protein band was similar to the mobility of theunprocessed form of wt BoNT/A. After optimization of expression, theBoNT/Aad^(ek) propeptide was highly enriched to virtual homogeneity fromSf-900 II medium in two steps: metal chelate affinity resin (FIG. 2A),followed by StrepTactin affinity chromatography (FIG. 2B).

The purified protein was then processed to the heterodimer by cleavagewith recombinant enterokinase. In the pilot reaction shown in FIGS.3A-B, the optimal enzyme/protein ratio for the cleavage was determinedHowever, it was also noted that an excess of enterokinase led tonon-specific protein degradation (FIG. 3B, lanes 4-7). This degradationcould be attributed either to secondary activity of the enterokinase, orto contaminants in the commercially available enterokinase preparations.The bulk of the expressed BoNT/Aad^(ek) propeptide was processed with0.007 units of enzyme per microgram of protein for 12 hours at 16° C.,and resulted in ˜95% completion of cleavage without visible degradationof light and heavy chains in the processed BoNT/Aad^(ek) heterodimer.Recombinant enterokinase was removed from the reaction mixture byincubation with Tag-off cleavage capture kit (Novagen).

To facilitate removal of the 10-His tag from the BoNT/Aad^(ek)propeptide, a TEV protease recognition sequence was introduceddownstream of the metal chelate affinity tag in the expressed protein.Due to the small size (19 aa) of the peptide released as a result of thecleavage, the shift in BoNT/Aad^(ek) propeptide mobility and the degreeof the enzymatic cleavage were not evident on the Coomassie-stainedgels. To examine and optimize conditions for TEV digest, a time coursepilot reaction was conducted. Samples of BoNT/Aad^(ek) without additionof the enzyme were used as a control. Aliquots from the reaction mixturewere taken at times ranging from 1 to 6 hours, separated by SDS PAGE,transferred to nitrocellulose membrane, and probed with anti His-tagmonoclonal antibody. The results are shown in FIG. 4. Incubation of onemicrogram of the BoNT/A1ad^(ek) with one unit of the AcTEV protease at30° C. for 6 hours led to almost complete removal of the His tag fromthe propeptide (FIG. 4, lane 10). The mobility of the propeptide band onSDS PAGE before and after the cleavage did not indicate any apparentnon-specific proteolytic activity associated with AcTEV. The AcTEVprotease was removed from the reaction mixture by affinitychromatography on TALON® resin.

Some recombinant proteins expressed in a secreted form in thebaculovirus system can be excessively glycosylated (Sydow et al.,“Overexpression of a Functional NMDA Receptor Subunit (NMDAR1) InBaculovirus-Infected Trichoplusia In Insect Cells,” Brain Res. Mol.Brain. Res. 41(1-2):228-240 (1996); Pechan et al., “HeterologousExpression of Maize (Zea mays L.) Mir1 Cysteine Proteinase in Eukaryoticand Prokaryotic Expression Systems,” Protein Expr. Purif. 34(1):134-141(2004), which are hereby incorporated by reference in their entirety).To rule out the presence of this post-translational modification, themobility of the expressed, processed, and denatured BoNT/Aad^(ek) aftertreatment with Endo-α-N-acetylgalactosaminidase and PNGaseF (New EnglandBiolabs, Cat #P0733S, P0704S) was compared with untreated samples. Nodifference in mobility of treated versus untreated samples were detectedby SDS PAGE.

The 12 aa S6 peptide tag placed downstream of the 10-His sequence andupstream of the N-terminus of the LC was incorporated as a target forSfp phosphopantetheinyl transferase in BoNT/Aad^(ek) and the otherderivatives described herein. Sfp phosphopantetheinyl transferasecatalyzes incorporation of small -molecule-CoA-based cargo to a specificserine residue within the S6 tag (Zhou et al., “Genetically EncodedShort Peptide Tags for Orthogonal Protein Labeling by Sfp and AcpSPhosphopantetheinyl Transferases,” ACS Chem. Biol. 2(5):337-346 (2007),which is hereby incorporated by reference in its entirety). As aprototypic cargo molecule, and to create a molecular probe to studyBoNT/A trafficking, a commercially available fluorescent conjugate ofCoA (CoA547, New England Biolabs) suitable for standard TAMRA and Cy3microscopy emission filter sets was used for BoNT/Aad^(ek) labeling.

A vector expressing recombinant C-terminally 6-His-tagged Sfpphosphopantetheinyl transferase from B. subtilis was expressed in E.coli, purified, and concentrated according to the cited procedure (Yinet al., “Site-Specific Protein Labeling by Sfp PhosphopantetheinylTransferase,” Nat. Protoc. 1(1):280-285 (2006), which is herebyincorporated by reference in its entirety). The original report, and NewEngland Biolabs protocols, described conditions used for in vitrolabeling of recombinant proteins expressed on the cell surface, i.e.,when complex mixtures of various biopolymers are present in thereaction. It was noted that under conditions tested, an increase of theCoA substrate concentration usually resulted in a higher background anddid not necessarily increase the signal to background ratio. Theaddition of fetal calf serum or 0.5% BSA to the reaction mixture reducedthe background staining.

During optimization of this site-specific BoNT/Aad^(ek) labeling invitro, background staining was also noticed. To minimize the background,and to avoid introducing contaminating proteins such as BSA into thelabeling reaction, BSA was replaced by a mixture of short peptides thatwere subsequently removed by dialysis and ultrafiltration. Thefluorescent derivative of the BoNT/Aad^(ek) heterodimer was synthesizedunder physiological conditions, as described supra. The results of theenzymatic labeling are shown in FIGS. 5A-B. In FIGS. 5A-B, labeled andunlabeled samples of BoNT/Aad^(ek) were separated on SDS PAGE and eitherstained with Coomassie, or transferred to a nitrocellulose membrane andscanned using a 532/580 nm excitation/emission filter set. Under thetested conditions, 7 ng of the fluorescently labeled BoNT/Aad^(ek) lightchain were visualized, an amount otherwise undetectable onCoomassie-stained gels. The recombinant Sfp phosphopantetheinyltransferase was removed from the reaction mixture by affinitychromatography on TALON® resin; the excess of CoA 547 and other lowmolecular weight components were removed by dialysis andultrafiltration.

N-Terminal Sequencing of BoNT/Aad^(ek)

BoNT/Aad^(ek) LC and HC, separated under reducing conditions on 10-14%Criterion SDS PAGE (Bio-Rad), were transferred to PVDF membrane(Bio-Rad, Cat. #162-0182) and stained with Bio-Safe Coomassie BB G-250(Bio-Rad, Cat. #161-0786). The proteins were subject to sequencing on anABI 494-HT Procise Edman Sequencer at the Molecular Structure Facilityat UC Davis. N-terminal sequencing of LC identified the first six aminoacids as GAGDSL (SEQ ID NO:37), and for the HC as ALNDLC (SEQ ID NO:38),which confirmed the predicted sequences of the protein N-termini.

Mass Spectrometric Analysis of BoNT/Aad^(ek) and CoA547-BoNT/Aad^(ek)

FIG. 6 shows an ESI Q-TOF MS/MS spectrum confirming the identity of theintact tryptic peptide from the C-terminus of BoNT/Aad^(ek) light chain,and confirming the sequence of the predicted enterokinase cleavage sitein the loop between LC and HC.

FIGS. 7A-C show MALDI-TOF MS (FIG. 7B) and ESI Q-TOF MS/MS (FIG. 7C)spectra of a tryptic peptide of sequence LLCVR (SEQ ID NO:39) from theBoNT/Aad^(ek) light chain linked via a disulfide bridge to a trypticpeptide of sequence ALNDLCIK (SEQ ID NO:40) from the BoNT/Aad^(ek) heavychain. The peptide of observed m/z 1489.84 was detected in the MALDI-TOFmass spectrum of the non-reduced toxin (FIG. 7B), which closely matchesthe predicted m/z of the singly charged monoisotopic dipeptide ion of1489.82. The peptides are absent from the MALDI-TOF MS spectrum of atryptic digest of the reduced BoNT/Aad^(ek) light chain (FIG. 7A). Thesequence of the peptide was confirmed by the MS/MS spectrum (FIG. 7C),and the peptide representing the heavy chain was identified by Mascotdatabase searching when including the mass of the disulfide bonded lightchain peptide.

After enzymatic labeling of BoNT/Aad^(ek) light chain by Sfpphosphopantetheinyl transferase with CoA 547, three separate in-geltryptic digests were analyzed by LC-MS in duplicate, and compared to theLC-MS spectra of digests of protein incubated with enzyme without CoA547 to estimate the labeling efficiency of the reaction. Because thelabeled N-terminal peptide was not observed by mass spectrometry,reduction of the amount of unlabeled peptide was used to estimatelabeling efficiency. Mean ion intensity of the unmodified N-terminalpeptide, after normalization to the total ion intensity of all observedtryptic peptides from each respective protein, was reduced by 69±6%compared to the intensity of the unmodified peptide.

Example 11 Expression, Purification, and Processing of BoNT/Aad^(tev)

The BoNT/Aad^(tev) construct is very similar to the BoNT/Aad^(ek)construct, but enables the heterodimer-forming cleavage step and removalof the 10-His tag to be performed during a single processing step withTEV protease. The design of this construct for expression in thebaculovirus system is provided in detail supra. The generation ofbaculovirus stock and the procedure for Sf9 infection and culture growthwas similar to BoNT/Aad^(ek), as described supra. The propeptide waspurified using the 2-step affinity chromatography procedure, and yieldedapproximately 30 mg per liter of insect cell culture. Removal of the10-His tag and processing of the propeptide were performedsimultaneously by incubating 1 μg of the BoNT/Aad^(tev) propeptide with2 units of AcTEV protease at 30° C. for 6 hours. Processed peptide wasseparated from AcTEV protease by affinity chromatography on TALON® resinand collected in flow-through fractions. Loss of protein during thisprocedure was ˜5%. FIGS. 8A-C show the purified protein separated by SDSPAGE under reducing and non-reducing conditions.

Example 12 Expression, Purification, and Processing of ΔLC-Peptide-BoNT/A^(tev) and ΔLC-GFP-BoNT/A^(tev)

The ΔLC constructs were developed to evaluate the role of the LC peptidein BoNT-mediated delivery to the neuronal cytosol, and the limits oncargo that can be targeted using HC-mediated mechanisms. The design ofconstructs for expression in the baculovirus system is provided indetail supra. The generation of baculovirus stock and the procedure forSf9 infection and culture growth was similar to the previous twoconstructs, except for differences in MOI and incubation time afterinfection, which were approximately 0.5 and 72 hours, respectively. Thepropeptides were purified using 2-step affinity chromatography, asdescribed supra. With both ΔLC derivatives, precipitation was notedduring the concentration step. To prevent protein precipitation, priorto the concentration step, TRITON® X-100 was added to filtered medium.TRITON® X-100 was present throughout the TALON® chromatographyprocedure, and was replaced with dialyzable n-octyl-β-D-glucopyranosidefor StrepTactin affinity chromatography. Removal of the 10-His tag andprocessing of the propeptides were performed simultaneously byincubating 1 μg of the propeptides with 2 units of AcTEV protease at 30°C. for 6 hours in the presence of 0.2% n-octyl-β-D-glucopyranoside,which was removed by dialysis and ultrafiltration after the cleavage.Processed peptides were separated from AcTEV protease by affinitychromatography on TALON® resin and collected in flow-through fractions.The yield of ΔLC-Peptide-BoNT/A^(tev) constituted approximately 2 mg perliter and ΔLC-GFP-BoNT/A^(tev) 1 mg per liter of insect cell culturemedium. FIGS. 8A-C show the purified proteins separated by SDS PAGEunder reducing and non-reducing conditions.

Example 13 Discussion of BoNT/A Derivatives that Retain Wild TypeFeatures Required for Native Trafficking

Botulinum neurotoxin type A (BoNT/A) is the most toxic protein known,with LD₅₀ values for mice of <1 pg/g. The consequent high riskassociated with handling large amounts of this toxin have hindered thestudy of BoNT/A absorption and trafficking in vitro and in vivo. Hightoxicity has also prevented use of quantities of toxin that can bedetected using standard protein protocols. Potential solutions to thischallenge include studying the protein domains expressed as separateentities, or studying atoxic versions of the full-length toxin, as, forexample, variants carrying point mutations that eliminate proteintoxicity associated with light chain metalloprotease. Because BoNTs arelarge (150 kDa), multi-domain, disulfide-bonded heterodimers, withmutual stabilization of the protein domains through multiple hydrogenbonds and hydrophobic interactions, it is challenging to producerecombinant BoNTs that retain all of the structural features andtrafficking properties of native BoNTs. Factors affecting the success ofprotein expression include the design of the expression construct andthe choice of expression system. When domains are expressed separately,they can be denatured, poorly soluble (Ahmed et al., “Light Chain ofBotulinum A Neurotoxin Expressed As an Inclusion Body From a SyntheticGene Is Catalytically and Functionally Active,” J. Protein Chem.19(6):475-487 (2000); Zhou et al., “Cloning, High-Level Expression,Single-Step Purification, and Binding Activity of His6-TaggedRecombinant Type B Botulinum Neurotoxin Heavy Chain Transmembrane andBinding Domain,” Protein Expr. Purif. 34(1):8-16 (2004); Lacy et al.,“Recombinant Expression and Purification of the Botulinum NeurotoxinType A Translocation Domain,” Protein Expr. Purif. 11(2):195-200 (1997),which are hereby incorporated by reference in their entirety), orunstable (Baldwin et al., “The C-Terminus of Botulinum Neurotoxin Type ALight Chain Contributes to Solubility, Catalysis, and Stability,”Protein Expr. Purif. 37(1):187-195 (2004), which is hereby incorporatedby reference in its entirety). BoNTs are also unusually sensitive tomechanical factors, and mild agitation has been reported to denature thenative toxin structure (Toth et al., “Extreme Sensitivity of BotulinumNeurotoxin Domains Towards Mild Agitation,” J. Pharm. Sci.98(9):3302-3311 (2009), which is hereby incorporated by reference in itsentirety). Even when the isolated domains are soluble and properlyfolded, their functionality in comparison with full-length native toxinis limited.

For these reasons, expression of mutated, atoxic forms of thefull-length botulinum neurotoxin A was pursued. The mature wt BoNT/A isa heterodimer formed between light (LC) and heavy (HC) chains, connectedvia a disulfide bridge; a second, intrachain disulfide bridge is alsopresent at the C-terminus of the toxin's receptor-binding domain(Krieglstein et al., “Covalent Structure of Botulinum Neurotoxin Type A:Location of Sulfhydryl Groups, and Disulfide Bridges and Identificationof C-Termini of Light and Heavy Chains,” J. Protein Chem. 13(1):49-57(1994), which is hereby incorporated by reference in its entirety). Anendogenous clostridial protease is involved in processing of wt BoNT/Apropeptide to the mature form. The cleavage occurs at a basic amino acidresidue in the loop between LC and HC. The recombinant BoNT/Aderivatives described herein were expressed as single chain propeptidesand were subsequently enzymatically processed to form LC-HCheterodimers, as in wt BoNT/A. However, the specificity of the cleavagewas increased by introducing a more complex sequence (>5 aa) into theloop between LC and HC as a protease recognition site.

In prior work (U.S. Patent Application Publication No. 2006/0204524 toIchtchenko et al.; Ichtchenko et al., “Full-Length Clostridium botulinumSerotype A Derivatives with Native Structure and Properties,” Neurotox.Res. 9:234 (2006), which are hereby incorporated by reference in theirentirety) expression of several full-length BoNT/A derivatives wasdescribed. The DNA construct encoding the first of these was generatedby consecutive subcloning of two phosphorylated linkers and five DNAfragments obtained from PCR reactions performed on genomic DNA isolatedfrom Clostridium botulinum A1 Hall strain. Although this derivative wasnot able to be expressed in E. coli, the baculovirus system enabled itsexpression in a secreted form that could be readily purified from theculture medium without harsh processing. The major problems encounteredin earlier baculovirus work were related to low yield of the proteinafter purification (approximately 0.35 mg per liter of insect cellculture medium) and the inability to establish a single steppurification procedure based on the original 6-His tag incorporated atthe N-terminus of the construct for this purpose. Either because ofinsufficient length, or partial burial of the tag in the globule of thelight chain, protein was eluted from the Ni²⁺ affinity resin in 40 mMimidazole along with multiple major contaminants. As a solution, a4-step conventional/affinity chromatography protocol was established.The complexity of the protocol contributed to the low yield of the finalproduct. These results were considered when designing and synthesizingthe constructs described herein.

The BoNT/A ad constructs reported here represent full length botulinumneurotoxin type A carrying two mutations, E₂₂₄>A and Y₃₆₆>A, introducedin the light chain. Both original amino acids, E₂₂₄ and Y₃₆₆, areconserved among different botulinum neurotoxin serotypes and are part ofthe catalytic core of the light chain metalloprotease responsible forcleavage of the substrate. The light chain in mutated full-length BoNT/Aad lacks the ability to cleave SNAP 25 (Rigoni et al., “Site-DirectedMutagenesis Identifies Active-Site Residues of the Light Chain ofBotulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun.288(5):1231-1237 (2001); Zhou et al., “Expression and Purification ofthe Light Chain of Botulinum Neurotoxin A: A Single Mutation AbolishesIts Cleavage of SNAP-25 and Neurotoxicity After Reconstitution With theHeavy Chain,” Biochemistry 34(46):15175-15181 (1995); Li et al.,“Probing the Mechanistic Role of Glutamate Residue In the Zinc-BindingMotif of Type A Botulinum Neurotoxin Light Chain,” Biochemistry39(9):2399-2405 (2000), which are hereby incorporated by reference intheir entirety). It was also shown that mutation of E₂₂₄ and Y₃₆₆ didnot change the secondary structure, topography of aromatic aminoresidues, Zn²⁺ content, or substrate binding ability of the LCmetalloprotease (Li et al., “Probing the Mechanistic Role of GlutamateResidue In the Zinc-Binding Motif of Type A Botulinum Neurotoxin LightChain,” Biochemistry 39(9):2399-2405 (2000), which is herebyincorporated by reference in its entirety). Several popular expressionsystems, including E. coli and Pichia pastoris, show high bias againstclostridial AT-rich DNA, resulting in slow growth (Baldwin et al., “TheC-Terminus of Botulinum Neurotoxin Type A Light Chain Contributes toSolubility, Catalysis, and Stability,” Protein Expr. Purif.37(1):187-195 (2004), which is hereby incorporated by reference in itsentirety), premature termination of protein synthesis, or initiation ofirrelevant translation from an alternative reading frame (Lacy et al.,“Recombinant Expression and Purification of the Botulinum NeurotoxinType A Translocation Domain,” Protein Expr. Purif. 11(2):195-200 (1997),which is hereby incorporated by reference in its entirety). Similarproblems were observed in attempts to express protein in E. coli, andthe initial attempts in the baculovirus expression system resulted inlow protein yields. Typically, the use of E. coli strains supplementedwith rare tRNAs, silent mutagenesis of the native clostridial DNA, orcodon-optimized synthetic DNA are used in attempts to resolve thisproblem (Ahmed et al., “Light Chain of Botulinum A Neurotoxin ExpressedAs an Inclusion Body From a Synthetic Gene Is Catalytically andFunctionally Active,” J. Protein Chem. 19(6):475-487 (2000); Sutton etal., “Preparation of Specifically Activatable Endopeptidase Derivativesof Clostridium botulinum Toxins Type A, B, and C and TheirApplications,” Protein Expr. Purif. 40(1):31-41 (2005), which are herebyincorporated by reference in their entirety). Here, a fully syntheticDNA sequence was created de novo, encoding full-length constructs thatwere optimized for expression in both Sf9 insect cells and E. coli, asexplained supra. A 7 aa spacer sequence was introduced, to separate theN-terminus of the light chain domain from the upstream sequence. In theconstructs described here the length of the metal affinity tag wasincreased from 6 to 10 histidine residues, which significantly improvedthe intended single step affinity purification using non-denaturingconditions.

The constructs described herein additionally contain an 8 aa StrepTag IIadded at their C-termini to improve the selectivity of purification.While the addition of extra amino acids to the C-terminus of theclostridial neurotoxins causes some concern for interference with theprotein binding to its cognate receptors, recent data obtained fromX-ray studies of a StrepTag II C-terminally fused BoNT/B in complex withits receptors showed that the amino acid sequence of this tag isspatially separated from the receptor-binding epitopes of BoNT/B (Jin etal., “Botulinum Neurotoxin B Recognizes Its Protein Receptor With HighAffinity and Specificity,” Nature 444(7122):1092-1095 (2006), which ishereby incorporated by reference in its entirety). Therefore, it wasdecided to use this tag in the design of the neurotoxins describedherein. The benefit of having both N- and C-terminally tagged protein isto allow separation of the full-length protein from N- or C-terminallytruncated products generated adventitiously in the host expressionsystem.

In early structural studies of BoNT/A isolated from Clostridiumbotulinum, it was shown that an endogenous protease, or trypsin, cancleave BoNT/A propeptide at K₄₄₄, followed by cleavage at K₄₄₈, whichgenerates the tetrapeptide G₄₄₅YNK₄₄₈ (SEQ ID NO:41) which is releasedas a cleavage product (DasGupta et al., “Botulinum Neurotoxin Type A:Sequence of Amino Acids at the N-Terminus and Around the Nicking Site,”Biochimie 72(9):661-664 (1990), which is hereby incorporated byreference in its entirety). In subsequent work, K₄₃₈ was identified asthe C-terminus of the mature LC, generated by the excision of the 10amino acid peptide, T₄₃₉KSLDKGYNK₄₄₈ (SEQ ID NO:42) from the wt BoNT/Aprecursor (Krieglstein et al., “Covalent Structure of BotulinumNeurotoxin Type A: Location of Sulfhydryl Groups, and Disulfide Bridgesand Identification of C-Termini of Light and Heavy Chains,” J. ProteinChem. 13(1):49-57 (1994), which is hereby incorporated by reference inits entirety). The most C-terminal amino acid of the light chain visiblein the published X-ray structure of the BoNT/A holotoxin is R₄₃₂ (Lacyet al., “Crystal Structure of Botulinum Neurotoxin Type A andImplications for Toxicity,” Nat. Struct. Biol. 5(10):898-902 (1998),which is hereby incorporated by reference in its entirety). There isthus a discrepancy between the native form of the light chain, extendingto position 438, and the form seen in the crystal structure, extendingonly to position 432. This discrepancy, which could influence BoNTproperties, could arise because the C-terminal residues beyond R₄₃₂ ofthe light chain in crystal are present, but are too flexible to generatedistinct electron density on X-ray. Alternatively, a trypsin-likeprotease may have cleaved the LC-HC propeptide at R₄₃₂ so that thisfragment is not actually present in the crystal structure. It wasrationalized that the constructs described herein should not beshortened by the length of the peptide loop excised from wt BoNT/Aprecursor because this might influence the 3D structural constraints onthis region, but should rather be made resistant to trypsin-likeproteases. Therefore, mutations K₄₃₈>H, K₄₄₀>Q, and K₄₄₄>Q wereintroduced into the constructs described herein to render the propeptidederivatives resistant to proteolytic cleavage by trypsin-like proteasesand to yield uniformly processed LC C-termini in the heterodimers.Trypsin treatment of wt BoNT/A can also lead to cleavage of itsreceptor-binding HC_(C) domain, and complete loss of toxicity (Shone etal., “Inactivation of Clostridium botulinum Type A Neurotoxin by Trypsinand Purification of Two Tryptic Fragments, Proteolytic Action Near theCOOH-Terminus of the Heavy Subunit Destroys Toxin-Binding Activity,”Eur. J. Biochem. 151(1):75-82 (1985), which is hereby incorporated byreference in its entirety). Therefore, the mutation 1(₈₇₁>N at theHC_(N)-HC_(c) junction was introduced into the constructs describedherein, rendering the HC insensitive to this type of non-specificcleavage as well (Chaddock et al., “Expression and Purification ofCatalytically Active, Non-Toxic Endopeptidase Derivatives of Clostridiumbotulinum Toxin Type A,” Protein Expr. Purif. 25(2):219-228 (2002);Shone et al., “Inactivation of Clostridium botulinum Type A Neurotoxinby Trypsin and Purification of Two Tryptic Fragments, Proteolytic ActionNear the COOH-Terminus of the Heavy Subunit Destroys Toxin-BindingActivity,” Eur. J. Biochem. 151(1):75-82 (1985), which are herebyincorporated by reference in their entirety).

The biological activity of botulinum neurotoxins requires proper foldingand disulfide bonding of the protein during post-translationalprocessing. There are 9 cysteine residues in wt BoNT/A, five of whichcarry free sulfhydryl groups and 4 of which are involved in formation oftwo disulfide bridges (Krieglstein et al., “Covalent Structure ofBotulinum Neurotoxin Type A: Location of Sulfhydryl Groups, andDisulfide Bridges and Identification of C-Termini of Light and HeavyChains,” J. Protein Chem. 13(1):49-57 (1994), which is herebyincorporated by reference in its entirety). Expression of individualBoNT/A domains with endogenous cysteines in the reduced state has beenreported to contribute to formation of protein aggregates (Baldwin etal., “The C-Terminus of Botulinum Neurotoxin Type A Light ChainContributes to Solubility, Catalysis, and Stability,” Protein Expr.Purif. 37(1):187-195 (2004); Lacy et al., “Recombinant Expression andPurification of the Botulinum Neurotoxin Type A Translocation Domain,”Protein Expr. Purif. 11(2):195-200 (1997), which are hereby incorporatedby reference in their entirety). The use of maltose-binding protein as afusion partner for expression of LC-HC_(N) clostridial polypeptides inE. coli has been beneficial for formation of S—S bonds in E. coli-basedexpression systems, albeit the nature of the bond(s) formed was notconfirmed (Sutton et al., “Preparation of Specifically ActivatableEndopeptidase Derivatives of Clostridium botulinum Toxins Type A, B, andC and Their Applications,” Protein Expr. Purif. 40(1):31-41 (2005),which is hereby incorporated by reference in its entirety).Maltose-binding protein probably not only contributes to solubility ofthe fusion polypeptides, but rather acts as a chaperone, promotingpartial export of the fusion protein into the oxidizing environment ofthe periplasm. A recent review describes a variety of methods tooptimize expression of cysteine-containing proteins in E. coli (deMarco, “Strategies for Successful Recombinant Expression of DisulfideBond-Dependent Proteins In Escherichia coli,” Microb. Cell. Fact. 8:26(2009), which is hereby incorporated by reference in its entirety). Thecurrently available reports of soluble, properly folded, andbiologically active, full-length clostridial polypeptides expressed inE. coli did not utilize strains that contain any of these specializedfeatures required for proper post-translational modifications (Rummel etal., “Two Carbohydrate Binding Sites in the HC_(c)-Domain of TetanusNeurotoxin Are Required for Toxicity,” J. Mol. Biol. 326(3):835-847(2003); Rummel et al., “The HC_(C)-Domain of Botulinum Neurotoxins A andB Exhibits a Singular Ganglioside Binding Site Displaying SerotypeSpecific Carbohydrate Interaction,” Mol. Microbiol. 51(3):631-643(2004); Rummel et al., “Synaptotagmins I and II Act as Nerve CellReceptors for Botulinum Neurotoxin G,” J. Biol. Chem.279(29):30865-30870 (2004); Bade et al., “Botulinum Neurotoxin Type DEnables Cytosolic Delivery of Enzymatically Active Cargo Proteins toNeurons Via Unfolded Translocation Intermediates,” J. Neurochem.91(6):1461-1472 (2004); Li et al., “Recombinant Forms of Tetanus ToxinEngineered for Examining and Exploiting Neuronal Trafficking Pathways,”J. Biol. Chem. 276(33):31394-31401 (2001), which are hereby incorporatedby reference in their entirety). Attempts to obtain full-lengthBoNT/Aad^(ek) in two different strains of E. coli have not beensuccessful. However, efforts with the baculovirus expression system havebeen successful on multiple levels, as described herein.

Four distinct BoNT/A derivatives were expressed and characterized usingthe baculovirus system. The first derivative, BoNT/Aad^(ek), representsthe substantially intact BoNT/A, with two mutations, E₂₂₄>A and Y₃₆₆>A,introduced to inactivate the toxin protease, and thereby render thederivative atoxic, and with an enterokinase site for processing theexpressed propeptide into the heterodimer This derivative was expressedas a soluble single chain protein secreted into insect cell culturemedium, and was purified to homogeneity by chromatography on metalaffinity resin and StrepTactin column, yielding 30 milligrams per literof culture medium. The propeptide was treated with AcTEV protease toremove the N-terminal 10-His tag, and was treated with enterokinase toproduce the LC-HC heterodimer The structural authenticity of theexpressed protein was confirmed by Western blotting with BoNT/Apolyclonal antibodies and by Edman degradation of LC and HC. MALDI-TOFand ESI-Q-TOF LC-MS/MS analysis of LC tryptic digests confirmeduniformity of the enterokinase cleavage at the LC-HC junction throughidentification of the predicted C-terminal peptide of the LC (FIGS.7A-C). The presence of the disulfide bridge formed between Cys₄₃₀ andCys₄₅₄ (aa numbers are from the sequence of wt BoNT/A) in the expressedprotein was confirmed by MALDI-TOF and ESI-Q-TOF LC-MS/MS analysisthrough identification of the S—S linked dipeptide in tryptic digests ofprocessed BoNT/Aad^(ek) heterodimer (FIG. 6).

The second derivative, BoNT/Aad^(tev) is similar to BoNT/Aad^(ek) butreplaces the enterokinase site between LC and HC with a TEV recognitionsequence. This avoids non-specific cleavage associated with excessenterokinase, and reduces the number of steps required for proteinprocessing. This derivative was also expressed as a soluble single chainprotein secreted into insect cell culture medium, and was purified tovirtual homogeneity by chromatography on metal affinity resin andStrepTactin column, yielding 30 milligrams per liter of culture medium.The propeptide was further processed with AcTEV protease tosimultaneously remove the N-terminal 10-His tag and to generate theLC-HC heterodimer. The structural authenticity of the expressedheterodimer, and formation of the S—S bridge between LC and HC in thisderivative, were confirmed by reduced and unreduced SDS PAGE and Westernblotting (FIGS. 8A-C).

In the third derivative, ΔLC-Peptide-BoNT/A^(tev), the entire catalyticdomain (P₂-F₃₉₀) of the light chain was removed, leaving a 54 aa LCsegment (N₃₉₁-G₄₄₅) that forms the disulfide bridge and otherwiseinteracts with the HC_(N). Two TEV protease cleavage sites in thisderivative allowed simultaneous removal of the N-terminal 10-His tag andprocessing of the precursor to generate the ΔLC-HC heterodimer. Thisderivative was expressed as a soluble single chain protein secreted intoinsect cell culture medium, but was found to have a tendency toprecipitate during concentration prior to the first affinitychromatography. To prevent precipitation, TRITON® X-100 was added to themedium during concentration and was maintained throughout processing.The protein was eluted from TALON® resin and purified further on aStrepTactin column in the presence of n-octyl-β-D-glucopyranoside. Afterprocessing with AcTEV protease to generate the ΔLC-HC heterodimer andremove the 10-His affinity tag, it became possible to remove detergentbecause the solubility of the processed heterodimer increased markedly.Yield of the protein was 2 milligrams per liter of culture medium. Thepurified AcTEV-processed protein migrated as ˜110 kDa band on SDS PAGEin the absence of reducing agent and, as expected in the presence ofβ-mercaptoethanol, separated as LC-remnant and HC with apparentmobilities of 10 and 100 kDa, respectively. Western blotting performedwith polyclonal antibodies raised against BoNT/A holotoxin confirmed thestructural identity of the ΔLC-Peptide-BoNT/A^(tev) heavy chain, but didnot detect the truncated LC (FIGS. 8A-C).

The fourth derivative, ΔLC-GFP-BoNT/A^(tev), originates from the thirdderivative by insertion of green fluorescent protein (GFP) into thesequence of the short LC remnant peptide at its N-terminus. It likewiseincludes the 54 aa LC segment (N₃₉₁-G₄₄₅) that forms the disulfidebridge and otherwise interacts with the HC_(N). Two TEV proteasecleavage sites in this derivative allow simultaneous removal of theN-terminal 10-His tag and processing of the precursor to the LC-HCheterodimer. Aggregation of this protein during concentration prior tochromatography also required use of a combination of detergents and thepurification scheme described supra for ΔLC-Peptide-BoNT/A^(tev). Yieldof the protein was 1 milligram per liter of culture medium. The purifiedAcTEV-processed protein migrated as ˜140 kDa band on SDS PAGE in theabsence of reducing agent and, as expected in the presence ofβ-mercaptoethanol, migrated as independent ΔLC-GFP and HC with apparentmobilities of 40 and 100 kDa, respectively. Western blotting performedwith polyclonal antibodies raised against BoNT/A holotoxin andmonoclonal antibody against GFP confirmed the structural identity ofΔLC-GFP -BoNT/A^(tev) light and heavy chains (FIGS. 8A-C).

The relatively low yield and aggregation of derivatives three and four,where the majority of the LC has been removed, is attributed toconformational instability and spatial tension in the expressedpropeptides. The published X-ray structure of BoNT/A holotoxin (Lacy etal., “Crystal Structure of Botulinum Neurotoxin Type A and Implicationsfor Toxicity,” Nat. Struct. Biol. 5(10):898-902 (1998), which is herebyincorporated by reference in its entirety) suggests that the N-terminalportion of the N₃₉₁-G₄₄₅ sequence is a flexible loop and not part of thedistinct protein fold. It was hypothesized that deletion of the entireLC catalytic domain exposes hydrophobic areas of the propeptide thatcannot optimally collapse due to the constraints imposed by thetethering of the loop on both ends, and that AcTEV cleavage relievesthis constraint by untethering one end so that the propeptide cancollapse into a more soluble conformation.

During early pilot stages using natural genes instead of the syntheticgenes reported here, to evaluate the utility of the baculovirusexpression system and the physiological activity of the designedconstructs, a full-length BoNT/A derivative without any introducedmutations that would inactivate the LC metalloprotease was expressed.When the non-mutated derivative was tested in vitro and in vivo, itdemonstrated toxicity and absorption kinetics comparable to that of thenative toxin. The expressed toxic derivative was tested in mouse phrenicnerve-hemidiaphragm preparations. Exocytosis was evoked by stimulationof the nerve trunk (0.2 Hz), and the muscle twitch was monitored. Whenadded at a concentration of ˜1×10⁻¹¹ M, the toxic derivative producedblockage of transmission in 167±17 min (n=4), comparable topharmaceutical preparations of BoNT/A. To insure that the blockade wasattributed to a botulinum toxin-type action, the derivative waspre-incubated (room temperature, 60 min) with rabbit antiserum raisedagainst the carboxy-terminal half of the native BoNT/A heavy chain(i.e., receptor-binding domain). When these experiments (n=3) wereperformed in the presence of this polyclonal BoNT/A antiserum, there wasno onset of paralysis throughout the duration of the experiment(approximately 400 minutes of tissue monitoring) (U.S. PatentApplication Publication No. 2006/0204524 to Ichtchenko et al.;Ichtchenko et al., “Full-Length Clostridium botulinum Serotype ADerivatives with Native Structure and Properties,” Neurotox. Res. 9:234(2006), which are hereby incorporated by reference in their entirety).

According to one embodiment, the recombinant BoNT/A derivativesdescribed herein carry an S6 tag in their N-terminal region. Specificconditions for in vitro CoA-fluorophore labeling were optimized andshown for BoNT/Aad^(ek). Prior studies using fluorescent-labeled BoNTsto evaluate either LC trafficking in neurons or BoNT/A uptake byepithelial or neuronal cells have primarily relied on two methods: (i)transient expression following transfection with LC sequences taggedwith a fluorescent marker (e.g., GFP) (Oyler et al., “Trafficking andPost-Translational Modifications of BoNT Light Chains Within Cells,”Abstracts of the 5^(th) International Conference on Basic andTheraupeutic Aspects of Botulinum and Tetanus Toxins, Denver, Colo. 24(2005); Fernandez-Salas et al., “Plasma Membrane Localization Signals Inthe Light Chain of Botulinum Neurotoxin,” Proc. Natl. Acad. Sci. (USA)101(9):3208-3213 (2004); Fernandez-Salas et al., “Is the Light ChainSubcellular Localization an Important Factor In Botulinum Toxin Durationof Action?” Mov. Disord. 19(Suppl 8):S23-S34 (2004), which are herebyincorporated by reference in their entirety), or (ii) attachment offluorescent tags to LC-HC BoNT heterodimers using chemical methods(Verderio et al., “Internalization and Proteolytic Action of BotulinumToxins in CNS Neurons and Astrocytes,” J. Neurochem. 73(1):372-379(1999); Lalli et al., “Functional Characterisation of Tetanus andBotulinum Neurotoxins Binding Domains,” J. Cell. Sci. 112(Pt16):2715-2724 (1999); Grumelli et al., “Internalization and Mechanism ofAction of Clostridial Toxins In Neurons,” Neurotoxicology 26(5):761-767(2005); Ahsan et al., “Visualization of Binding and Transcytosis ofBotulinum Toxin by Human Intestinal Epithelial Cells,” J. Pharmacol.Exp. Ther. 315(3):1028-1035 (2005), which are hereby incorporated byreference in their entirety). The transient expression system providesimportant information on LC interactions after the recombinant proteinaccumulates in neurons, but cannot be used to explore the normaltrafficking route that delivers LC to the neuronal cytoplasm. Thechemical method for attaching probes is limited by the lack of selectivefluorophore attachment (e.g., both LC and HC will be modified), and theinevitable formation of complex mixtures (unlabeled, singly, andmultiply labeled species). The latter problem makes it particularlydifficult to confidently attribute behavior of fluorescently labeledBoNT molecules as being representative of native trafficking andbiological activity; the most intensely fluorescently labeled moleculescould produce the most intense signal despite potentially being theleast related to native trafficking, while a small population ofunmodified molecules could be responsible for any observed biologicalactivity. One study showed that a protein with relatively simple spatialorganization, such as ˜50 kDa glutathione S-transferase (GST), can lose90% of its activity as a result of chemical labeling, while targetedenzymatic incorporation of a fluorescent probe into GST completelypreserved its biological function (Taki et al.,“Transglutaminase-Mediated N- and C-Terminal Fluorescein Labeling of aProtein Can Support the Native Activity of the Modified Protein,”Protein Eng. Des. Sel. 17(2):119-126 (2004), which is herebyincorporated by reference in its entirety). In the original workdescribing the design and selection of the S6 tag for site-selectiveenzymatic attachment of fluorophores to recombinant proteins, theligand-binding properties of two enzymatically labeled proteins, EGF andtransferrin receptors, were tested. The data confirmed thatsite-specific labeling of the expressed receptors with CoA-fluorophoredid not interfere with physiological binding or recycling of either EGFor transferrin (Zhou et al., “Genetically Encoded Short Peptide Tags forOrthogonal Protein Labeling by Sfp and AcpS PhosphopantetheinylTransferases,” ACS Chem. Biol. 2(5):337-346 (2007); Yin et al.,“Site-Specific Protein Labeling by Sfp Phosphopantetheinyl Transferase,”Nat. Protoc. 1(1):280-285 (2006); Zhou et al., “An Eight ResidueFragment of an Acyl Carrier Protein Suffices for Post-TranslationalIntroduction of Fluorescent Pantetheinyl Arms In Protein Modification Invitro and In vivo,” J. Am. Chem. Soc. 130(30):9925-9930 (2008), whichare hereby incorporated by reference in their entirety).

One aspect of the present invention provides experimental evidencedemonstrating enzymatic site-specific fluorescent labeling of arecombinant full-length BoNT/A derivative, with the fluorophore attachedto a specific site located on the N-terminal peptide, designed as suchto minimally interfere with native BoNT structure. Interestingly, theability to incorporate CoA 547 into recombinant protein also indirectlyconfirms the sorting mechanism through which these derivatives aresecreted into insect culture medium. Sfp phosphopantetheinyl transferaseis a ubiquitous enzyme that is present in the cytoplasm of insect cells,and would therefore be expected to attach endogenous CoA to the BoNTderivatives described herein if they were not secreted into the culturemedium and thereby separated from the endogenous phosphopantetheinyltransferase in the insect cell cytoplasm. The unmodified serine residuewithin the S6 tag identified by Edman degradation confirms that therecombinant protein undergoes a sorting mechanism that protects it fromenzymatic action of the endogenous cytoplasmic Sfp phosphopantetheinyltransferase in the Sf9 cell expression host. According to massspectrometric analysis of CoA547-modified versus non-modifiedderivative, the fluorescent marker was attached to approximately 69±6%of the BoNT/Aad^(ek) added to the enzymatic labeling reaction mixture.With the existing commercially available derivatives, such as CoA-biotin(New England Biolabs, Cat. #S9351S), it is possible to achieve 100%probe incorporation by streptavidin affinity enrichment. The chemistrybehind coupling of various moieties to CoA has been described (Yin etal., “Site-Specific Protein Labeling by Sfp PhosphopantetheinylTransferase,” Nat. Protoc. 1(1):280-285 (2006), which is herebyincorporated by reference in its entirety) and can provide almostunlimited opportunities in terms of coupling small molecules, bioactivepeptides, and peptidomimetics to the atoxic BoNT/A derivatives describedherein, exemplifying a versatile platform for targeting diversetherapeutic agents to the neuronal cytoplasm.

Three previously mentioned reports describe expression and purificationof recombinant, atoxic, full-length BoNT derivatives, with pointmutations to the LC active site (Kiyatkin et al., “Induction of anImmune Response by Oral Administration of Recombinant Botulinum Toxin,”Infect. Immun. 65:4586-4591 (1997); Pier et al., “Recombinant HolotoxoidVaccine Against Botulism,” Infect. Immun. 76(1):437-442 (2008); Webb etal., “Production of Catalytically Inactive BoNT/A1 Holoprotein andComparison With BoNT/A1 Subunit Vaccines Against Toxin Subtypes A1, A2,and A3,” Vaccine 27(33):4490-4497 (2009), which are hereby incorporatedby reference in their entirety). All three of these recombinantholotoxoids were developed with the specific intention of producing arecombinant BoNT vaccine, rather than with the intention of developingprobes for BoNT trafficking studies, or delivery vehicles that cantarget the neuronal cytosol. The recombinant metalloprotease-inactivatedBoNT/C reported by Kiyatkin et al., “Induction of an Immune Response byOral Administration of Recombinant Botulinum Toxin,” Infect. Immun.65:4586-4591 (1997), which is hereby incorporated by reference in itsentirety, was non-toxic at doses up to 10 μg per mouse. BoNT/A^(RYM)described by Pier et al., “Recombinant Holotoxoid Vaccine AgainstBotulism,” Infect. Immun. 76(1):437-442 (2008), which is herebyincorporated by reference in its entirety showed no apparent toxicity upto doses of 1 μg per mouse, and ciBoNT/A HP described by Webb et al.,“Production of Catalytically Inactive BoNT/A1 Holoprotein and ComparisonWith BoNT/A1 Subunit Vaccines Against Toxin Subtypes A1, A2, and A3,”Vaccine 27(33):4490-4497 (2009), which is hereby incorporated byreference in its entirety, was non-toxic at doses up to 50 μg per mouse,indicating that the respective holotoxoids can elicit immunity at thenon-toxic doses mentioned supra. Although data on toxicity at higherdoses was not included in these reports, the relative absence oftoxicity in different vaccine candidates might be considered somewhatsurprising if in fact the recombinant proteins follow the native BoNTtargeting pathway. BoNT/A LC mutated in its active site has been shownin crystallographic studies to still be capable of binding its substrateSNAP-25 (Breidenbach et al., “Substrate Recognition Strategy forBotulinum Neurotoxin Serotype A,” Nature 432:925-929 (2004), which ishereby incorporated by reference in its entirety), and if delivered toneurons at high doses might be expected to bind and potentiallyinterfere with the exocytotic machinery at high doses. Secondly, HCsdelivered to the endosomal compartment of motor neurons might beexpected to retain their pore-forming capability in the acidic endosomalenvironment, and likewise potentially disrupt physiologic neuronalactivity (Shone et al., “A 50-kDa Fragment From the NH2-Terminus of theHeavy Subunit of Clostridium botulinum Type A Neurotoxin Forms ChannelsIn Lipid Vesicles,” Eur. J. Biochem. 167(1):175-180 (1987), which ishereby incorporated by reference in its entirety). Since the mutated LCdoes not disable the apparatus for its own endocytosis, theserecombinant holotoxoids should accumulate in neurons when administeredat high concentrations, and might be expected to have some effect onneuronal function. It should be noted in this context that immunogenicactivity can be retained in the absence of structural features requiredfor native BoNT trafficking. Thus, although it is clear that theserecombinant holotoxoids are effective vaccine candidates and that theirlow toxicity may provide the acceptable therapeutic index required, thedata presented do not address the question of whether the recombinantholotoxoids follow the native BoNT trafficking pathway, and therefore donot currently support their potential utility for BoNT traffickingstudies, or as delivery vehicles that can target the neuronal cytosol.

The above data demonstrate that the baculovirus expression system, usedin conjunction with synthetic gene constructs described herein, enablesproduction of a series of atoxic, full-length, and truncated BoNTderivatives that preserve important structural features of native BoNT.The derivatives can be recovered from culture media as solubledisulfide-bonded heterodimers, and can be purified to homogeneity usingtwo-stage, non-denaturing, and highly selective affinity purification.The ability to recover the expressed derivatives as soluble proteinsobviates the need to recover insoluble expressed derivatives frominclusion bodies using denaturing conditions. Retaining native BoNTstructure during harsh solubilization and purification steps ischallenging. Renaturation is equally challenging, because of the large,complex, disulfide-bonded structure of BoNTs. Attempts to restoredisulfide bonding in recombinant BoNT/A HC_(C) domain expressed in P.pastoris, which is only ⅓ of the full-length molecule, illustrate thedifficulty of achieving physiologically relevant protein folding duringrecombinant protein expression (Bouvier et al., “Identifying andModulating Disulfide Formation In the Biopharmaceutical Production of aRecombinant Protein Vaccine Candidate,” J. Biotechnol. 103(3):257-271(2003), which is hereby incorporated by reference in its entirety).Another recent publication highlights vulnerability of botulinumneurotoxins to rapid and irreversible denaturation during handling, aseven mild agitation was shown to alter the secondary structure of HCsand LCs from diverse BoNT serotypes (Toth et al., “Extreme Sensitivityof Botulinum Neurotoxin Domains Towards Mild Agitation,” J. Pharm. Sci.98(9):3302-3311 (2009), which is hereby incorporated by reference in itsentirety). The sensitivity of BoNT/A to isolation and purificationconditions is also reflected in the wide batch-to-batch variabilityobserved during pharmaceutical BoNT/A production from clostridialcultures in terms of BoNT/A specific activity units per mg oftherapeutic protein preparation. Because the expression and purificationmethodology employed in the present invention circumvents all types ofdenaturing conditions, the expressed BoNT derivatives described hereinhave been designed to retain native BoNT structure to a greater extentthan methods requiring exposure to harsh reagents.

Example 14 BoNT/Aad^(ek) is Non-Toxic to Neuronal Cultures and CanEffectively Compete with wt BoNT/A

To confirm that BoNT/A ad is non-toxic, but otherwise hascharacteristics similar to wt BoNT/A, primary rat spinal cord cells wereexposed to as much as 500 nM BoNT/A ad. In particular, these primary ratspinal cord neurons were used to determine whether BoNT/Aad^(ek) itselfhas any observable effects on cultured neuronal cells, and whether ithas the ability to compete with, or otherwise antagonize, the effects ofwt BoNT/A on the neuronal cultures. This assay can detect toxicity offemtomolar amounts of wt BoNT/A (measured by SNAP 25 cleavage) (Pellettet al., “A Neuronal Cell-based Botulinum Neurotoxin Assay for HighlySensitive and Specific Detection of Neutralizing Serum Antibodies,” FEBSLett 581(25): 4803-08 (2007); Pellett et al., “Comparison of the PrimaryRat Spinal Cord Cell (RSC) Assay and the Mouse Bioassay for BotulinumNeurotoxin Type A Potency Determination,” J. Pharmacol. Toxicol. Methods61(3):304-310 (2010), which are hereby incorporated by reference intheir entirety), yet incubation of cells with 500 nM BoNT/A ad resultedin no detectable BoNT-specific proteolytic activity (FIG. 10). In otherwords, exposure of cells to as much as 500 nM BoNT/Aad^(ek) did notresult in detectable cleavage of intracellular SNAP 25, the target ofthe wt toxin.

Structural and partial functional identity of wt BoNT/A and BoNT/A adwas further confirmed by the dose-dependent ability of BoNT/A ad toblock SNAP 25 cleavage by wt BoNT/A (FIG. 10). In this competitionassay, pre-incubation of the primary rat spinal cord cells with 500 nMBoNT/Aad^(ek) blocked intracellular SNAP 25 cleavage induced by 0.5 nMwt BoNT/A.

Example 15 Toxicity Studies of BoNT/Aad^(ek)

To examine toxicity in vivo, the LD₅₀ of BoNT/A ad was determined bymouse bioassay, and was approximately 50 μg/kg intraperitoneally, whichis about 100,000-fold higher than the LD₅₀ of wt BoNT/A.

The wt BoNT/A is targeted to the neuromuscular junction where it cleavesSNAP 25 and causes neuromuscular paralysis by disabling the machinery ofregulated exocytosis. Specificity of BoNT/A ad binding to thepresynaptic sites at the neuromuscular junction (“NMJ”) was confirmed byimmunocytochemical analysis of triangularis sterni nerve-musclepreparations after in vivo injection of mice (FIGS. 11A-C). Theseresults indicate that residual toxicity of BoNT/A ad is associated withNMJ-specific accumulation.

Next, it was determined if the light chain of BoNT/A ad undergoestranslocation to the neuronal cytosol. Rat hippocampal neuronal cultures(Vicario, “Long-term Culture of Hippocampal Neurons,” in CurrentProtocols in Neuroscience Suppl. 26: 3.2.1.-3.2.13 (John Wiley & Sons,Inc. 2004), which is hereby incorporated by reference in its entirety)were exposed to BoNT/A ad, and analyzed by immunostaining using anantibody, MAb F1-40, which is a well characterized BoNT/A L-chainspecific antibody (Stanker et al., “Development and PartialCharacterization of High-affinity Monoclonal Antibodies for BotulinumToxin Type A and Their Use in Analysis of Milk by Sandwich ELISA,” J.Immunol. Methods 336 (1):1-8 (2008); Scotcher et al., “EpitopeCharacterization and Variable Region Sequence of F1-40, a High-AffinityMonoclonal Antibody to Botulinum Neurotoxin Type a (Hall strain)” PLoSOne 4 (3):e4924 (2009), which are hereby incorporated by reference intheir entirety) with high specificity, sensitivity, and reproducibilityin immunocytochemistry.

Immunostaining of neurons with three different treatment and chaseregimens of BoNT/A ad is shown in FIG. 12. BoNT/A ad uptake could bevisualized when neuronal cultures were exposed to BoNT/A ad for 30-90minutes at 37° C. as an extracellular punctuate pattern (FIG. 12,Columns B and D, Row 1), representing the active synaptic contacts andpoints of BoNT/A ad entry at the axon termini. This staining graduallydisappears during the chase (FIG. 12, Column D, Rows 2 and 3), due tocontinuous intracellular uptake of the extracellular BoNT/A ad andabsence of additional recombinant protein in the medium. When cells arechased for 90 min after incubation with BoNT/A ad (FIG. 12, Row 3), mostof the LC ad staining is concentrated intracellularly, adjacent to theplasma membrane and co-localized with SNAP 25. This type of staining hasnever been shown for wt BoNT/A because the amount of wt LC delivered tothe neuronal cytoplasm is exceedingly low (below the level forvisualization). The immunostaining of the neurons shown in Row 3 of FIG.12 is consistent with earlier reports where LC/A-GFP constructs wereexpressed in neuronal and nonneuronal cultures. The pattern of theLC/A-GFP distribution after transfection shows that intrinsic propertiesof LC/A contributed to accumulation of LC/A-GFP on the inner leaflet ofthe neuronal plasma membrane after expression (Fernández-Salas et al.,“Is the Light Chain Subcellular Localization an Important Factor inBotulinum Toxin Duration of Action?” Mov. Disord., 19, Suppl 8: 23-34(2004); Wang et al., “Novel Chimeras of Botulinum Neurotoxins A and EUnveil Contributions From the Binding, Translocation, and ProteaseDomains to Their Functional Characteristics” J. Biol. Chem.283(25):16993-17002 (2008); Tsai et al., “Targeting Botulinum NeurotoxinPersistence by the Ubiquitin-proteasome System,” Proc. Natl. Acad. Sci.USA., 107 (38):16554-16559 (2010), which are hereby incorporated byreference in their entirety).

It was then directly examined whether LC ad interacted with SNAP 25 inthe cytosol of neuronal cells exposed to BoNT/A ad. As shown in FIGS.13A and 13B, the ˜52 kDa band corresponding to LC ad was co-precipitatedwith an anti-SNAP 25 antibody. Neither SNAP 25 nor LC ad were detectedin a control experiment with BHK fibroblasts treated with BoNT/A adunder the same conditions. If LC ad binds SNAP 25 in the neuronalcytoplasm without cleaving it, then binding could lead to sequestrationof SNAP 25 from the tripartite complex and disruption of neuronalactivity. This would suggest that the toxicity of BoNT/A ad at highdoses was a result of increased LC ad accumulation in the neuronalcytosol due to unlimited internalization—a feature beneficial forneuronal delivery of a wide range of therapeutic moieties. Therefore, itwas examined whether exocytosis was affected by exposure of neuronalcells to BoNT/A ad.

Rat hippocampal neurons were pre-loaded under depolarizing conditionswith FM 143 dye. When 100-500 nM BoNT/A ad was added to the depolarizingmedium, an inhibition of exocytosis (fusion of FM-143 labeled synapticvesicles with plasma membrane) occurred in a dose-dependent manner.Similar results were obtained for wt BoNT/A, albeit the testing wasperformed with picomolar concentrations of the toxin. Finally, theabsorption profile of a BoNT derivative modified through enzymaticlipidation was assessed (FIGS. 14A-B). Lipidation should result in rapidbinding to cell membranes, thereby reducing diffusion. The ΔLC-GFPBoNT/Aderivative is readily taken up by neuronal cells due to the presence ofBoNT/A heavy chain which binds to the neuron-specific SV2 receptor. Innon-neuronal cells, however, which are deficient in SV2 (such as COS7 orBHK), there is no specific binding. For lipidation, the ΔLCGFP-BoNT/Aderivative was labeled enzymatically with palmitoyl-CoA. Non-neuronalCOS7 cells were used to assess lipid-mediated binding in the absence ofreceptor-meditated binding.

Incubation of COS7 cells for various times with ΔLC-GFP-BoNT/A did notproduce any visible pattern of protein uptake. In comparison, when thepalmitoylated derivative of ΔLC-GFPBoNT/A was added to the cells, within5 minutes the staining of plasma membrane was evident. Longer incubationwith palmitoylated ΔLC-GFPBoNT/A resulted in intracellular uptake of theprotein consistent with endosome/lysosome internalization similar toother studies with lapidated GFP derivatives (Antos et al., “LipidModification of Proteins Through Sortase-Catalyzed Transpeptidation,” J.Am. Chem. Soc. 130 (48):16338-16343 (2008), which is hereby incorporatedby reference in its entirety).

Prophetic Example 16 Formation of BoNT/Aad Derivatives to which LipidMoieties Have Been Selectively Incorporated at the S6 Cargo Site, withthe Purpose of Improved Targeting of BoNT Action, and MinimizingUnintended Diffusion and Pharmacologic Action at Unintended Sites

BoNT/Aad, atoxic derivative retains the essential wild type toxinfeatures required for native trafficking, but has been rendered atoxicthrough the introduction of metalloprotease-inactivating mutations inthe light chain of BoNT/A. To produce a pharmaceutically active BoNT/Aderivative, the inactivating point mutaions can be restored to theirnative sequence. Both such toxic and atoxic derivatives carry an S6peptide tag upstream of the linker sequence adjacent to the N-terminusof the light chain, which allows site-specific enzymatic attachment ofvarious molecules for potential use in therapeutic intervention.

Here, a method is described to selectively incorporate lipid moietiesinto recombinant BoNT/A derivatives by enzymatic coupling to a cargoattachment peptide (e.g., the S6 peptide tag), in order to restrict thediffusion of the protein adduct from the site of injection. The goal ofthis project is to test a novel approach for precisely localizing thepharmaceutical action of lipidated BoNT/Aad within the targetedneuromuscular junction at the site of injection, thereby preventingeffects associated with unintended dispersal and spread of the toxinbeyond the site of application.

To accomplish this, the derivative will be additionally modified tocontain a sequence specifically cleaved by the BACE-1 enzyme(B-secretase) specifically localized to the surface of neurons, insertedbetween the spacer sequence and S6 cargo attachment site. This enablesthe BoNT/A derivative to be specifically released from its attachment atthe external surface of the plasma membrane of neurons, and thereby toimprove localization of its pharmacologic action to neurons at the siteof application. As an example, lipidated ΔLC-GFP-BoNT/A ad, describedabove, can be used to study the association of the protein with cellsthrough the lipid tail and/or through receptor-mediated endocytosis viathe HC of BoNT/A ad. To study the fate of the internalized protein(light chain) after translocation from an endocytic comparment to thecytoplasmic comparment of neurons, see infra, lipidated BoNT/A adderivatives will be used, because the ΔLC-GFP-BoNT/A ad derivative willremain stuck in the endocytic compartment due to the rigidity of the GFPportion of ΔLC-GFP-BoNT/A ad.

Expression and purification of atoxic BoNT/A derivative with cleavagerecognition sequence positioned between the S6 tag and the spacersequence upstream of the N-terminus of BoNT/Aad will be performed asdescribed supra. Removal of the 10-His purification tag and processingof BoNT/Aad single chain propeptide to heterodimer will be performedwith TEV protease, as described supra.

Labeling of BoNT/Aad with palmitoyl-CoA will be performed withrecombinant Sfp phosphopantetheinyl transferase from B. subtilis asdescribed supra. Yield of palmitoylated BoNT/Aad will be evaluated bymass-spectral analysis. Purification of palmitoylated protein fromunmodified BoNT/Aad will be performed by fractionation with Triton X-114with modification: Instead of ion exchange chromatography, eitheraffinity chromatography on StrepTactin agarose or hydrophobicchromatography on octyl-sepharose will be used.

The length and type of lipid moiety attached to the protein not onlycontributes to protein diffusibility and kinetics of absorption, but tothe route of internalization and fate of internalized proteins in vitro(Antos et al., “Lipid Modification of Proteins Through Sortase-CatalyzedTranspeptidation,” J. Am. Chem. Soc. 130 (48):16338-16343 (2008), whichis hereby incorporated by reference in its entirety). Therefore, inaddition to generating palmitoylated, aliphatic (C-16) adduct of BoNT/Aad derivative, an adduct will be created with a longer aliphatic chain(C -22) and an adduct will be created where cholesterol will be used asa lipid moiety. These adducts will be obtained by modifying purifiedBoNT/A ad protein (Band et al., “Recombinant Derivatives of BotulinumNeurotoxin A Engineered for Trafficking Studies and Neuronal Delivery,”Protein Expr. Purif. 71(1):62-73 (2010), which is hereby incorporated byreference in its entirety) through Sfp phosphopantetheinyl transferaselabeling (Zhou et al., “Genetically Encoded Short Peptide Tags forOrthogonal Protein Labeling by Sfp and AcpS PhosphopantetheinylTransferases,” ACS Chem. Biol. 2 (5):337-346 (2007), which is herebyincorporated by reference in its entirety) with lipidated CoAprecursors. The precursors will include commercially available(palmitoyl-CoA, C-16, Sigma-Aldrich) and synthesized (C-22 aliphatic-CoA or cholesterol-CoA, Irvine Chemistry Laboratory) compounds.

A test will be done to compare the diffusion pattern and effects oflocal and systemic distribution of BoNT/Aad and palmitoylated BoNT/A ad(or other lipidated BoNT/A ad noted above) with targeted solubilityafter injection into mouse hindlimb muscle.

It is expected that limited diffusion of palmitoylated BoNT/Aad from thesite of injection will be observed, in comparison with unpalmitoylatedderivative, as has been described for other in vitro lipidated proteins.

Mice will be used as an animal model, because their small muscle sizeincreases the sensitivity of immunoassays. Swiss-Webster adult male CD1mice weighing ˜30 g will be used for injection. Mice will be housed ingroups of six and food and water will be provided ad libitum. Mice willbe maintained on a 12-h light/dark photoperiod for 4 days before thestart of experiments. All work with animals will be performed bypersonnel trained in the safe and humane use of laboratory animalsaccording to existing and pending animal protocols.

Animals (time-pregnant rats) are used as a source for producing adherentcultures of primary rat hippocampal neurons, embryonic spinal cord cells(BACE1-positive), and fetal myoblast cultures (BACE1-negative) as amodels for the type of cells encountered after administration of thederivatives in vivo. Live mice will also be used to test toxicity,diffusion, and systemic distribution of pairs of nonlipidated/lipidatedBoNT/A ad variants in order to identify a lead candidate for futuredevelopment of the pharmacologically active recombinant version ofBoNT/A, obtained by reversion of the inactivating mutations. Primarycell cultures: Rat embryonic hippocampal neurons and spinal cord cellsrepresent the widely in vitro used system to dissect physiologicalmechanism of BoNT/A trafficking and internalization. Live timed-pregnantrats will be used as a source of the primary neuronal cultures. Thoughestablished immortalized cell lines are available with some neuron-likefeatures, their exclusive use for studies of BoNT/A ad derivativestrafficking is undesirable due to the up/down regulation and mutationsin multiple gene products involved in the BoNT pathway. The requirementfor primary neuronal culture for these types of studies is widelyrecognized as the standard approach. All work with animals will beperformed by personnel trained in the safe and humane use of laboratoryanimals according to existing and pending animal protocols.

For in vitro studies, while adherent cultures of primary rat hippocampalneurons (Vicario-Abejon, “Long-term Culture of Hippocampal Neurons,” inCurrent Protocols in Neuroscience, Suppl. 26, 3.2.1.-3.2.13 (John Wiley& Sons, Inc., 2004), which is hereby incorporated by reference in itsentirety), embryonic spinal cord cells (Pellett et al., “A NeuronalCell-based Botulinum Neurotoxin Assay for Highly Sensitive and SpecificDetection of Neutralizing Serum Antibodies,” FEBS Lett., 581(25):4803-4808 (2007); Pellett et al. “Comparison of the Primary RatSpinal Cord Cell (RSC) Assay and the Mouse Bioassay for BotulinumNeurotoxin Type A Potency Determinatio,” J. Pharmacol. Toxicol. Methods61(3):304-310 (2010), which are hereby incorporated by reference intheir entirety) (BACE1-positive), and fetal myoblast cultures (Pin etal., “Embryonic and Fetal Rat Myoblasts Express Different PhenotypesFollowing Differentiation in vitro,” Dev. Genet. 14 (5):356-368 (1993);Pin et al., “Embryonic and Fetal Rat Myoblasts Form Different MuscleFiber Types in an Ectopic in vivo Environment,” Dev. Dyn. 224(3):253-266 (2002), which are hereby incorporated by reference in theirentirety) (BACE1-negative) will be used as models of the most abundantnerve and muscle tissue at the site of injection in vivo, some assayswill rely on use of neuronal cell lines (such as PC 12 or Neuro 2A,BACE1-positive) or non-neuronal cell lines (such as COS7 or BHK,BACE1-negative) in suspension cultures.

The first step will be to measure protein association with the cellmembrane and to assess resistance to diffusion. To measure relativeability to associate with cells, lipidated and nonlipidatedΔLC-GFP-BoNT/A derivatives will be added to cells suspended inserum-free medium, and the extent to which the presence of the lipidtail and differences in the lipid moiety contribute to binding of theproteins to cells will be quantified. Membrane binding kinetics will bemeasured by comparing fluorescence of the medium vs. fluorescence of thecells as described (Antos et al., “Lipid Modification of ProteinsThrough Sortase-Catalyzed Transpeptidation,” J. Am. Chem. Soc. 130(48):16338-16343 (2008), which is hereby incorporated by reference inits entirety). It is expected that the majority of lipidatedΔLC-GFPBoNT/A derivatives will be rapidly and effectively bound by allcells through insertion of the lipid moiety into the plasma membrane.

Diffusibility will be measured in monolayers of cells grown on anitrocellulose matrix. ΔLC-GFPBoNT/A or/and BoNT/A ad derivatives willbe added in millicell inserts (Millipore) positioned in the center ofthe growth circle. The diffusibility of the proteins will be directlymeasured by visualizing and quantifying the diameter of greenfluorescence and its intensity relative to the diameter of the insert(0% diffusibility) and the diameter of the plate (100% diffusibility) asdescribed (Flaumenhaft et al., “Heparin and Heparan Sulfate Increase theRadius of Diffusion and Action of Basic Fibroblast Growth Factor,” J.Cell. Biol., 111 (4):1651-1659 (1994), which is hereby incorporated byreference in its entirety). Those lipidated BoNT derivatives thatexhibit a ≧90% reduction in diffusibility compared to their nonlipidatedcounterparts will be selected for further characterization.

Possible cytotoxic effects of the BoNT/A ad and lipidated BoNT/A ad willbe determined, as described in Francis et al., “Enhancement ofDiphtheria Toxin Potency by Replacement of the Receptor Binding Domainwith Tetanus Toxin C-fragment: a Potential Vector for DeliveringHeterologous Proteins to Neurons,” J. Neurochem. 74 (6):2528-2536(2000), which is hereby incorporated by reference in its entirety. Thecytotoxicity will be evaluated over a concentration range from 1 to 500nM. Cultures will also be observed microscopically for signs of toxicityup to 96 hours post-exposure. Cultures exposed to wt BoNT/A anduntreated cells will be included as controls.

Comparison of protein uptake in neuronal and non-neuronal cultures willalso be conducted. An attractive feature of the lipidated BoNTderivatives is the possibility of their efficient uptake and degradationby non-neuronal cells, which would abet removal of excess toxin. It isknown that the type of lipid tail affects protein internalization. Antoset al., “Lipid Modification of Proteins Through Sortase-CatalyzedTranspeptidation,” J. Am. Chem. Soc. 130 (48):16338-16343 (2008), whichis hereby incorporated by reference in its entirety. To ensure lysosomaltargeting of the lipidated BoNT/A ad in non-neuronal cells,internalization patterns of different lipidated BoNT/A derivatives willbe compared using (a) immunocytochemical staining and (b) subcellularfractionation of the treated cells, followed by Western blotting. Therate of uptake relative to nonlipidated BoNT/A ad will be calculated.The internalization of the proteins by cells in culture will be assessedat different time points by two methods: first, by comparison of theintensity of LC ad signal on Western blot with antibodies against LC,and second, by LC ad association with specific cellular markers, such asSNAP 25 (cytoplasmic target for LC ad binding), Rab5/EEA1 (earlyendosome marker), Rab7 (late endosome marker), or LAMP1 (lysosomalmarker) as described in de Araujo et al., “Isolation of EndocyticOrganelles by Density Gradient Centrifugation,” Methods Mol. Biol.424:317-33 (2008); Huber et al., “Organelle Proteomics: Implications forSubcellular Fractionation in Proteomics,”Circ. Res. 92:962-968 (2003);Lemichez et al., “Membrane Translocation of Diphtheria Toxin Fragment AExploits Early to Late Endosome Trafficking Machinery,” Mol. Microbiol.23:445-457 (1997); and Ratts et al., “The Cytosolic Entry of DiphtheriaToxin Catalytic Domain Requires a Host Cell Cytosolic TranslocationFactor Complex,” J. Cell Biol. 160:1139-1150 (2003), which are herebyincorporated by reference in their entirety.

Additional evidence of LC ad presence in the neuronal cytoplasm will beperformed by co-immunoprecipitation from cultures with anti-SNAP 25antibodies, as described in Example 15 (FIGS. 13A-C). As many as 3 pairsof nonlipidated/lipidated BoNT constructs will be tested. Those thatexhibit, relative to BoNT/A ad, a 90% reduction in diffusibility and a10-fold increased rate of absorption by cells in culture, and which aretargeted to a lysosomal degradation pathway in non-neuronal cells but tophysiological LC ad translocation to the cytoplasm in neuronal cells,will be tested further.

The experimental outcome of the studies related to BoNT/A diffusion fromthe site of injection are not expected to be in agreement with commonperceptions based on the principle that larger proteins diffuse moreslowly through an identical aqueous medium compared with smallerproteins. According to this principle, it would be predicted that BoNTsof greater size or molecular weight will be less likely to diffuseoutside the target tissue compared with those of smaller size. Thus, inthis view, BOTOX®, composed of uniform 900 kDa complexes, should be lesslikely to diffuse outside the target tissue compared with DYSPORT® (aheterogeneous mixture of 500-900 kDa complex sizes) or XEOMIN® (pure 150kDa toxin). In multiple studies it was found that BoNT/A injectedintramuscularly exhibited some diffusion to muscles adjacent to the siteof injection, and when the same amount of active neurotoxin was used,regardless of manufacturer or average MW, the observed effect wasindistinguishable. However, the volume of injection and the total amountof injected protein are factors affecting both local and systemic spreadof the toxin. Therefore, for the planned experiments, the same amount ofeither palmitoylated or non-palmitoylated BoNT/Aad will be used forinjection.

The BoNT/Aad and palmitoylated BoNT/Aad protein concentration will bedetermined by the BCA-protein assay kit (Pierce), equalized andreconstituted with 0.9% sodium chloride to a final concentration 0.4mg/ml. Before injection, mice will be anesthetized. The injection volumewill be 25 μl. The protein will be injected in the tibialis anteriormuscle in one hindlimb while carrier alone (control) will be injected inthe contralateral muscle.

The extent of diffusion of BoNT/Aad versus palmitoylated BoNT/Aad willbe evaluated by examining the direct pattern of protein immunostainingand by the effect of BoNT on muscles located at different distances fromthe site of injection. In particular, sections of the soleus muscle,which is close to the injected tibialis anterior muscle, thegastrocnemius muscle, which is next to the soleus, and the even moredistant quadriceps femoris muscle, located in a rostral position, willbe evaluated.

Lethal doses of BoNT/A released to circulation cause death fromrespiratory arrest resulting from neuromuscular paralysis produced byaccumulation of toxin in the phrenic nerve. Therefore, potentialdispersal of the toxin through the circulation following injection intotibialis anterior muscle will be assessed by immunostaining of thediaphragm with antibodies against BoNT/A holotoxin.

According to a previous study, the most notable changes in diffusion ofthe toxin from the site of injection occurred within 1-48 hours afterinjection. Therefore, the immunostaining pattern of BoNT/Aad diffusionwill be studied after injection within this time frame.

The expression of N-CAM in mouse hindlimb muscles at different timesfollowing injection will be used as a readout of the effect generated byBoNT/Aad. N-CAM can be detected with high sensitivity and spatialresolution by histological and Western blot analyses. N-CAM is presenton the surface of embryonic myotubes, but it is lost as developmentproceeds. N-CAM is nearly absent from adult muscle, but muscledenervation induces the reappearance of N-CAM. Paralysis of skeletalmuscle by BoNT/A is sufficient to activate N-CAM expression. However,the action of the toxin, stemming from accumulation of its light chainin the cytosol of affected neurons, causes significantly delayedreappearance of N-CAM relative to a rapidly changed BoNT/A diffusionpattern. N-CAM is usually detected within 5-30 days after injection.This same time frame will be used.

Immunostaining will be performed 1, 2, 6, 12 hours, and one and two daysafter injection (for direct BoNT/A immunodetection) and 2, 7, 14, 30,and 60 days after injection (for N-CAM immunostaining). Tibialisanterior, soleus, gastrocnemius, and quadriceps femoris muscles will bedissected from both hindlimbs of the mice and frozen in isopentaneprecooled in liquid nitrogen. Preparation of mouse diaphragm will beperformed in situ. Muscle cryosections (10 μm) will be probed withpolyclonal antibodies against BoNT/A holotoxin (Staten Serum Institut,Denmark) diluted 1:1000, or polyclonal antibodies to N-CAM (Chemicon)diluted 1:500 in blocking solution. Goat anti-rabbit IgG conjugated toAlexa-555 (Invitrogen) diluted 1:200 in blocking solution will be usedfor detection. Alexa-488 alpha-bungarotoxin will be used forcounterstaining of NMJ. Images will be collected using a confocalmicroscope.

The presence of BoNT/Aad and the expression level of N-CAM in differentmuscles after injection with either form of BoNT/Aad will also beassessed quantitatively by Western blot analysis. The resulting datawill be normalized using tubulin as a loading standard and will beplotted as intensity units relative to the basal value of thenon-injected muscle.

After dissection, tissue lysates will be prepared for analysis byWestern blot. Samples will be loaded (10 μg/lane) on 4%-12% Tris-HClpolyacrylamide gels (Bio-Rad) and subjected to electrophoresis. Theproteins will be transferred to nitrocellulose membranes (Bio-Rad), andprobed either with polyclonal antibodies against BoNT/A holotoxin(Staten Serum Institut, Denmark) diluted 1:5000 or polyclonal antibodiesto N-CAM (Chemicon) diluted 1:300 in TBST supplemented with 5% nonfatmilk. Membranes will also be probed with anti-tubulin monoclonalantibody (1:1,000, Sigma Aldrich), which will serve as an internalstandard for protein quantification. Densitometric analysis will beperformed using Typhoon image analysis software (GE Healthcare).

The immunostaining panels and bars on quantification plots will berepresentative of several sections/protein extract preparations (n=4-6)made from each of the different injected animals (n=4). Descriptivestatistics of means and standard deviations (±SD) will be calculated.The t-test will be used post-hoc to determine significance ofdifferences.

Another preferred way to analyze the diffusion of pairs ofnonlipidated/lipidated BoNT/A ad variants is to examine the presence ofthe protein by ELISA. The double sandwhich ELISA for wt BoNT/A is moresensitive that direct immunostaining and is preferable. The localdiffusion of BoNT/A ad will be evaluated by assessing presence of BoNTby immunoreactivity in three distal muscle groups: the soleus muscle,which is close to the injected tibialis anterior muscle, thegastrocnemius muscle, which is distal to the soleus, and the more distalquadriceps femoris muscle, located in a rostral position. The percentreduction in diffusibility will be calculated both by distance (musclegroup) and by quantity (ELISA signal).

For evaluation of systemic spread of BoNT, the immunoreactivity will betested by ELISA in phrenic nerve preparations (the phrenic nerve isknown to be a target of wt BoNT/A in circulation), the tibialis anteriormuscle of the other (noninjected) limb, and in serum. Samples for ELISAwill be prepared as described in Whelchel et al., “Molecular Targets ofBotulinum Toxin at the Mammalian Neuromuscular Junction,” Mov. Disord.19, Suppl 8: S7-S16 (2004), which is hereby incorporated in itsentirety. The most notable changes in diffusion of wt toxin from thesite of injection occur within 1-48 hours after injection (Tang-Liu etal., “Intramuscular Injection of ¹²⁵I-botulinum Neurotoxin-complexVersus ¹²⁵I-Botulinum-free Neurotoxin: Time Course of TissueDistribution,” Toxicon 42 (5): 461-469 (2003), which is herebyincorporated by reference in its entirety). Therefore, tissue will becollected for analysis at six time points 1-48 hours after injection.The results will be normalized against tissue from animals injected withvehicle only.

Percent reduction in systemic exposure will be calculated from ELISAdata as rate of appearance and quantity in phrenic nerve and serum. 5mice are expected to be used per time point, as was used previously(Carli et al., “Assay of Diffusion of Different Botulinum NeurotoxinType A Formulations Injected in the Mouse Leg,” Muscle Nerve 40 (3):374-380 (2009), which is hereby incorporated by reference in itsentirety).

With the control group, the projected number of animals for the pair oflipidated/nonlipidated BoNT/A ad derivatives will be 120. Descriptivestatistics of means and standard deviations (±SD) will be calculated.The t-test will be used post-hoc to determine significance ofdifferences. Limited diffusion of lipidated BoNT/A ad is expected fromthe site of injection in comparison with nonlipidated derivatives, ashas been described for other in vitro lipidated proteins (Antos et al.,“Lipid Modification of Proteins Through Sortase-CatalyzedTranspeptidation,” J. Am. Chem. Soc. 130 (48):16338-16343 (2008); Groganet al., “Synthesis of Lipidated Green Fluorescent Protein and itsIncorporation in Supported Lipid Bilayers,” J. Am. Chem. Soc. 127 (41),14383-14387 (2005), which are hereby incorporated by reference in theirentirety).

This experiment will investigate a novel approach for preciselylocalizing the pharmaceutical action of botulinum neurotoxin A (BoNT/A)(e.g., BOTOX®, DYSPORT®) within the targeted neuromuscular junction atthe site of injection, thereby preventing effects associated withunintended dispersal and spread of the toxin beyond the site of entry.BoNT/A therapy has a good safety record, which depends partly on thetoxin's ability to remain relatively localized at the site of injection.The ability of the toxin to spread from the site of the injection todistant sites is a consequence of the fact that the toxin is soluble inaqueous solution and can be transported from the site of injectionthrough the circulation or diffused locally to nearby tissue. Safetyconcerns are likely to become more important as increasing BoNT/A dosesare used to treat conditions such as cerebral palsy or spasticity. Inthis regard, symptoms of generalized weakness have been described inBoNT-treated patients. These concerns are clearly valid, sincetherapeutic use of BoNT resulted in 28 deaths between 1989 and 2003,while the number of therapeutic applications of the toxin is rapidlyincreasing.

The low toxicity of BoNT/Aad makes it an ideal candidate for traffickingstudies, because the amount of protein used in vivo can provide reliableand direct immunodetection, a goal which has never been accomplishedusing wt BoNT/A because of its extremely high toxicity. In addition, theability to selectively incorporate lipidated moieties into recombinantBoNT/Aad by enzymatic coupling can restrict the diffusion of the proteinadduct from the site of injection. Both of these properties—low toxicityand the ability to enzymatically modify the recombinant protein will beused to advantage.

It is anticipated that palmitoylated BoNT/Aad will be targeted forinsertion into the plasma membrane of cells proximal to the injectionsite. Thus, palmitoylated BoNT/Aad will be excluded from the circulationimmediately after injection, and its capacity for systemic dispersalwill be minimal An additional important modification of the expressedBoNT/Aad will be a 13 amino acid sequence, SEISY↓EVEFRWKK (SEQ IDNO:43), which will be positioned between the cargo attachment peptidesquence and the spacer sequence upstream of the N-terminus of therecombinant protein. This peptide provides the specific substratesequence for BACE1, an aspartic acid membrane-bound protease involved inthe pathology of Alzheimer's disease, and which is predominantlyexpressed in cells of neural origin. Among non-neuronal cells at thesite of injection, the internalization of palmitoylated BoNT/Aad shouldresult in non-specific uptake of the recombinant palmitoylated protein.In neurons, in contrast, because of the presence of BACE1 on the cellsurface, recombinant BoNT/Aad should be cleaved, releasing solubledichain BoNT/Aad in the proximity of synaptic contact. Releasedrecombinant BoNT/Aad should enter the cell via the normal, heavychain-mediated, double receptor mechanism of internalization followed bytranslocation of the light chain into the neuronal cytosol.

Prior to incorporation of the canonical BACE1 recognition sequence intoexisting BoNT/A ad constructs (as mentioned above), a cell-based FRETassay will be used along with a variety of commercially availableFRET-based BACE1 substrates (Sigma, Invitrogen, Siena Biotech, etc.)(Gruninger-Leitch et al., “Substrate and Inhibitor Profile of BACE(beta-secretase) and Comparison with Other Mammalian AsparticProteases,” J. Biol. Chem., 277 (7):4687-4693 (2002), which is herebyincorporated by reference in its entirety) to optimize the BACE1recognition sequence for the greatest cleavage efficiency in neuronalcells and minimal/absent cleavage in non-neuronal cells. The amino acidsequence of the substrate that best fits these criteria will beincorporated in BoNT/A ad as an alternative to the canonical sequence.To estimate the efficiency of BACE1-mediated cleavage afterinternalization of lipidated BoNT/A ad, adducts will also be used inwhich the lipid moiety is C13-labeled. Three BoNT/A ad constructs willbe used to compare the relative yield of LC ad internalized throughBoNT/A-receptor mediated endocytosis, which should result in LC adtranslocation into the cytoplasm: nonlipidated BoNT/A ad, lipidatedBoNT/A ad, and lipidated BACE1-BoNT/A ad. Cytosolic fractions fromneuronal cultures exposed to all three types of proteins will beprepared by digitonin solubilization, as described in Example 15 (FIGS.13A-C). LC ad from these fractions will be co-immunoprecipitated withanti-SNAP 25 antibodies, separated on SDS PAGE and the resulting Westernblot will be probed with anti-LC MAb F1-40 as described (FIGS. 13A-C).It is expected that there will be comparable signals from LC ad from thecultures treated with BoNT/A ad and lipidated BACE1-BoNT/A ad, butminimal or no signal from lipidated BoNT/A ad, which should remainanchored to the membrane. It is also expected that LC ad from thefraction obtained after treatment with lipidated BACE1-BoNT/A ad willlack the radioactive lipid moiety as a consequence of BACE1-mediatedcleavage.

This project is intended to complete preliminary steps in thepre-clinical development of a non-diffusible botulinum neurotoxin Aformulation with targeted solubility, which is intended as a therapeuticfor neurological disorders.

Although the invention has been described in detail for the purposes ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

What is claimed:
 1. An isolated Clostridium botulinum neurotoxinpropeptide comprising: a light chain region; a heavy chain region,wherein the light and heavy chain regions are linked by a disulfidebond; an intermediate region connecting the light and heavy chainregions and comprising a highly specific protease cleavage site, whereinsaid highly specific protease cleavage site has three or more specificadjacent amino acid residues that are recognized by the highly specificprotease in order to enable cleavage; and a cargo attachment peptidesequence to enable site-specific attachment of cargo, wherein the cargoattachment peptide sequence is positioned upstream of the light chainregion and is separated from the N-terminus of the light chain region byan amino acid spacer sequence.
 2. The propeptide according to claim 1,wherein the Clostridium botulinum is serotype A.
 3. The propetideaccording to claim 1, wherein the cargo attachment peptide sequencecomprises an S6 peptide sequence of SEQ ID NO:2.
 4. The propeptideaccording to claim 1 further comprising: a signal peptide coupled to thecargo attachment peptide sequence, wherein the signal peptide issuitable to permit secretion of the neurotoxin propeptide from aeukaryotic cell to a medium.
 5. The propeptide according to claim 4further comprising: a 10-His affinity tag positioned between andconnecting the signal peptide to the cargo attachment peptide sequence.6. The propeptide according to claim 5 further comprising: a TEVrecognition sequence positioned between and connecting the 10-Hisaffinity tag to the cargo attachment peptide.
 7. The propeptideaccording to claim 5 further comprising: an 8 amino acid StrepTag IIconnected to the propeptide at the C-terminus.
 8. The propeptideaccording to claim 1, wherein the highly specific protease cleavage siteis selected from an enterokinase cleavage site and a TEV recognitionsequence.
 9. The propeptide according to claim 1, wherein the light andheavy chain regions are not truncated.
 10. The propeptide according toclaim 1, wherein the entire catalytic domain of the light chain regionhas been removed.
 11. The propeptide according to claim 10 furthercomprising: a fluorophore connected to the N-terminus of the light chainregion.
 12. The propeptide according to claim 1, wherein the cargo isselected from the group consisting of lipid moieties, therapeuticagents, marker molecules, and targeting agents.
 13. The propeptideaccording to claim 12, wherein the cargo is a lipid moiety selected fromthe group consisting of fatty acids, neutral lipids, phospholipids, andcomplex lipids.
 14. The propeptide according to claim 12, wherein thecargo is a lipid moiety selected from the group consisting ofpalmitoyl-CoA, C-22 aliphatic CoA, and cholesterol CoA.
 15. Thepropeptide accoding to claim 12, further comprising a neuron-specificprotease cleavage site positioned between the N-terminus of the lightchain region and the cargo.
 16. The propeptide according to claim 1,wherein the amino acid spacer sequence comprises at least 7 amino acidresidues.
 17. An isolated, physiologically active Clostridium botulinumneurotoxin produced by cleaving the propeptide according to claim 1 atthe highly specific protease cleavage site, wherein the light chainregion and the heavy chain region are linked by a disulfide bond. 18.The isolated Clostridium botulinum neurotoxin according to claim 17,wherein the Clostridium botulinum is serotype A.
 19. The isolatedClostridium botulinum neurotoxin according to claim 17 furthercomprising: a signal peptide coupled to the cargo attachment peptidesequence, wherein the signal peptide is suitable to permit secretion ofthe neurotoxin from a eukaryotic cell to a medium.
 20. The isolatedClostridium botulinum neurotoxin according to claim 19 furthercomprising: a 10-His affinity tag positioned between and connecting thesignal peptide to the cargo attachment peptide sequence.
 21. Theisolated Clostridium botulinum neurotoxin according to claim 20 furthercomprising: a TEV recognition sequence positioned between and connectingthe 10-His affinity tag to the cargo attachment peptide sequence. 22.The isolated Clostridium botulinum neurotoxin according to claim 17further comprising: an 8 amino acid StrepTag II connected to theneurotoxin at the C-terminus.
 23. The isolated Clostridum botulinumneurotoxin according to claim 18 further comprising: E₂₂₄>A and Y₃₆₆>Amutations.
 24. The isolated Clostridum botulinum neurotoxin according toclaim 18 further comprising: one or more mutations in the light chainregion selected from K₄₃₈>H, K₄₄₀>Q, and K₄₄₄>Q.
 25. The isolatedClostridium botulinum neurotoxin according to claim 18 furthercomprising: a K₈₇₁>N mutation in the heavy chain region.
 26. Theisolated Clostridium botulinum neurotoxin according to claim 17, whereinthe light and heavy chain regions are not truncated.
 27. The isolatedClostridium botulinum neurotoxin according to claim 17, wherein theentire catalytic domain of the light chain region has been removed. 28.The isolated Clostridium botulinum neurotoxin according to claim 27further comprising: a fluorophore connected to the N-terminus of thelight chain region.
 29. The isolated Clostridium botulinum neurotoxinaccording to claim 17, wherein the cargo is selected from the groupconsisting of lipid moieties, therapeutic agents, marker molecules, andtargeting agents.
 30. The isolated Clostridium botulinum neurotoxinaccording to claim 17, wherein the neurotoxin is atoxic.
 31. Theisolated Clostridium botulinum neurotoxin according to claim 18, havinga structure selected from BoNT/Aad^(ek), BoNT/Aad^(tev),ΔLC-Peptide-BoNT/A^(tev), and ΔLC-GFP-BoNT/A^(tev).
 32. The isolatedClostridium botulinum neurotoxin according to claim 17, wherein thecargo attachment peptide sequence comprises at least 7 amino acidresidues.
 33. The isolated Clostridium botulinum neurotoxin according toclaim 17, wherein the neurotoxin (a) has an LD₅₀ that is at least75,000-fold higher than the LD₅₀ of wild-type Clostridium botulinumneurotoxin and/or (b) accumulates within neuronal cytosol in higheramounts than wild type Clostridium botulinum neurotoxin.
 34. Thepropeptide according to claim 1, wherein the propeptide has an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:6.